Compositions, formulations and interleukin production and purification

ABSTRACT

Described herein are cholix-IL-10 fusion proteins, and methods of use thereof, which can be characterized by a distinct response in an individual when administered. This distinct response can comprise changes in levels of one or more markers in the individual and/or co-localization of IL-10 in the lamina propria of the individual. Further described herein, in some embodiments, are oral formulations of the cholix-IL-10 fusion proteins. Described herein are methods for the purification of an IL-10 delivery construct, including methods for refolding and enrichment, which can result in maintenance of a high percentage of the IL-10 delivery constructs in the biologically active dimer form. Described herein are oral formulations configured for site-specific release of a therapeutic protein in the small intestines or colon. In some cases, the therapeutic protein is in the form of a dimer, such as an IL-10 delivery construct capable of crossing the gut epithelium.

CROSS-REFERENCE

This application is a continuation application of U.S. application Ser.No. 17/169,390, filed Feb. 5, 2021, which is a continuation applicationof International Patent Application No. PCT/US2020/046545, filed Aug.14, 2020, which claims the benefit of U.S. Provisional Application No.62/888,144, filed Aug. 16, 2019, U.S. Provisional Application No.62/888,237, filed Aug. 16, 2019, U.S. Provisional Application No.62/986,579, filed Mar. 6, 2020, U.S. Provisional Application No.62/887,963, filed Aug. 16, 2019, U.S. Provisional Application No.62/887,933, filed Aug. 16, 2019, U.S. Provisional Application No.62/898,934, filed Sep. 11, 2019, U.S. Provisional Application No.62/971,126, filed Feb. 6, 2020, U.S. Provisional Application No.62/898,709, filed Sep. 11, 2019, U.S. Provisional Application No.62/898,729, filed Sep. 11, 2019, U.S. Provisional Application No.62/939,495, filed Nov. 22, 2019, U.S. Provisional Application No.62/970,627, filed Feb. 5, 2020, U.S. Provisional Application No.63/020,996, filed May 6, 2020, U.S. Provisional Application No.63/033,077, filed Jun. 1, 2020, U.S. Provisional Application No.62/898,899, filed Sep. 11, 2019; U.S. Provisional Application No.63/013,309, filed Apr. 21, 2020; U.S. Provisional Application No.62/986,557 filed Mar. 6, 2020; and U.S. Provisional Application No.63/055,886, filed Jul. 23, 2020; which applications are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 12, 2020, isnamed 40566-721_601_SL.txt and is 729,437 bytes in size.

BACKGROUND OF THE INVENTION

While oral administration can be a convenient and desirable route forthe administration of protein pharmaceuticals, challenges presented bythis administration route include the acidic environment of the stomach,which can cause denaturation of protein structure, including dimers, andhydrolysis of chemical bonds, variable pH across various regions of thegastrointestinal tract, and the presence of proteolytic enzymes whichare secreted into the GI tract and break down proteins into smallerfragments. Furthermore, even if protein pharmaceuticals are able tosurvive these challenges and arrive intact in the lower GI tract, it canbe difficult for such pharmaceuticals to cross the intestinal epitheliumdue to their large size.

Additionally, some therapeutic proteins are active (or more active) inthe dimer form. Thus, their therapeutic utility may be compromised whenproduced or formulated in a manner that does not result in properdimerization. Common purification and processing protocols may preventthe desired dimer formation, resulting in (for example) an excessivelyhigh proportion of monomers or aggregates.

SUMMARY OF THE INVENTION

Described herein, in certain embodiments, are delivery constructsconsisting of an amino acid sequence set forth in SEQ ID NO: 5 or SEQ IDNO: 13. In some embodiments, the delivery construct consists of theamino acid sequence set forth in SEQ ID NO: 5. In some embodiments, thedelivery construct consists of the amino acid sequence set forth in SEQID NO: 13. In some embodiments, the delivery construct is part of ahomodimer. In some embodiments, the delivery construct is disposedwithin a composition that is formulated for oral administration, whereinthe composition for oral administration comprises a plurality ofdelivery constructs identical to the delivery construct, and wherein atleast 80% of the delivery constructs are in a dimer form.

Described herein, in certain embodiments, are methods of treating aninflammatory disease in a subject, the method comprising administeringto the subject an effective amount of a delivery construct as describedherein. In some embodiments, the inflammatory disease is ulcerativecolitis, proctitis, pouchitis, Crohn's disease, multiple sclerosis (MS),systemic lupus erythematosus (SLE), graft versus host disease (GVHD),rheumatoid arthritis, inflammatory bowel disease (IBD), Celiac disease,psoriatic arthritis, or psoriasis. In some embodiments, the inflammatorydisease is ulcerative colitis. In some embodiments, the inflammatorydisease is Crohn's disease. In some embodiments, the inflammatorydisease is celiac disease.

Described herein, in certain embodiments, are methods of refolding anIL-10 delivery construct, the method comprising: (i) contactinginclusion bodies comprising the IL-10 delivery construct with asolubilization solution comprising a chaotropic agent to produce asoluble IL-10 delivery construct; (ii) contacting the soluble IL-10delivery construct with a refolding solution comprising reducedglutathione and oxidized glutathione to produce a refolded IL-10delivery construct; wherein the method does not comprise contacting thesoluble IL-10 delivery construct with a sulfitolysis agent or a reducingagent prior to the contacting of step (ii). In some embodiments, theIL-10 delivery construct comprises a carrier. In some embodiments, thecarrier is derived from a polypeptide secreted by a bacterium. In someembodiments, the bacterium is Vibrio cholerae. In some embodiments, thepolypeptide secreted by Vibrio cholerae is a cholix polypeptide. In someembodiments, the IL-10 delivery construct has at least 80%, 85%, 90%,92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an aminoacid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL-10delivery construct has a V1L substitution at amino acid position 1 ofthe carrier.

In some embodiments, the refolding solution comprises a ratio of thereduced glutathione to the oxidized glutathione from 0.8:1 to 1.2:1. Insome embodiments, the refolding solution comprises from 0.75 mM to 1.5mM reduced glutathione. In some embodiments, the refolding solutioncomprises from 0.25 mM to 0.75 mM oxidized glutathione. In someembodiments, the refolding solution has a pH from 7.5 to 8.5. In someembodiments, the refolding solution comprises arginine, Tris, and EDTA.In some embodiments, the refolding solution comprises sucrose. In someembodiments, the refolding solution comprises arginine, sucrose, Tris,EDTA, or a combination thereof. In some embodiments, arginine is presentin the refolding solution at a concentration of between 900 mM and 1.1M. In some embodiments, sucrose is present in the refolding solution ata concentration of between 200 mM and 300 mM. In some embodiments, Trisis present in the refolding solution at a concentration of from 75 mM to125 mM. In some embodiments, EDTA is present in the refolding solutionat a concentration of from 1.75 mM to 2.25 mM.

In some embodiments, the method further comprises lysing a cellcomprising the inclusion bodies. In some embodiments, the cell is abacterium. In some embodiments, the bacterium is Escherichia coli. Insome embodiments, the lysing comprises high-pressure homogenization. Insome embodiments, the method further comprises isolating the inclusionbodies. In some embodiments, the chaotropic agent comprises guanidinehydrochloride or urea. In some embodiments, the solubilization solutionfurther comprises Tris. In some embodiments, the contacting the solubleIL-10 delivery construct with a refolding solution occurs for at least16 hours. In some embodiments, the contacting the soluble IL-10 deliveryconstruct with a refolding solution occurs from 12 hours to 18 hours.

In some embodiments, the contacting the IL-10 delivery construct with arefolding solution occurs from 2° C. to 8° C. In some embodiments, themethod further comprises a first sterile filtration of the refoldedIL-10 delivery construct. In some embodiments, the first sterilefiltration occurs after the contacting with the refolding solution. Insome embodiments, the method further comprises performing a tangentialflow filtration of the refolded IL-10 delivery construct. In someembodiments, the tangential flow filtration comprises diafiltration. Insome embodiments, the diafiltration comprises a first diavolume, asecond diavolume, a third diavolume, and a fourth diavolume. The In someembodiments, the first diavolume and the second diavolume comprise acold buffer. In some embodiments, the third diavolume and the fourthdiavolume comprise a room temperature buffer. In some embodiments, thecold buffer and the room temperature buffer comprise Tris and NaCl.

Described herein, in certain embodiments, are methods of enriching forIL-10 delivery construct dimers from a pool comprising IL-10 deliveryconstructs in a dimer form, a monomer form, and an aggregate form, themethod comprising: (i) performing anion exchange (AEX) chromatography onthe pool by binding the IL-10 delivery construct dimers to an anionexchange column and subsequently eluting the IL-10 delivery constructdimers from the anion exchange column, thereby creating a firstplurality of fractions, one of which is a first fraction enriched inIL-10 delivery constructs in the dimer form; and (ii) performing ceramichydroxyapatite (CHT) chromatography on the fraction enriched in IL-10delivery constructs in the dimer form, thereby creating a secondplurality of fractions, one of which is a second fraction furtherenriched in IL-10 delivery constructs in the dimer form. In someembodiments, the IL-10 delivery construct comprises a carrier. In someembodiments, the carrier is derived from a polypeptide secreted by abacterium. In some embodiments, the bacterium is Vibrio cholerae. Insome embodiments, the polypeptide secreted by Vibrio cholerae is acholix polypeptide. In some embodiments, the IL-10 delivery constructhas at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence set forth in SEQ ID NO: 5.In some embodiments, the IL-10 delivery construct has a V1L substationat amino acid position 1 of the carrier. In some embodiments, the methodfurther comprises determining a percentage of IL-10 delivery constructsin the dimer form in each fraction of the first plurality of fractions.In some embodiments, the determining is by size exclusionchromatography. In some embodiments, the size exclusion chromatographyis size exclusion high performance liquid chromatography (SE-HPLC). Insome embodiments, the method further comprises determining a percentageof IL-10 delivery constructs in the dimer form in each fraction of thesecond plurality of fractions. In some embodiments, the determining isby size exclusion chromatography. In some embodiments, the sizeexclusion chromatography is size exclusion high performance liquidchromatography (SE-HPLC). In some embodiments, at least 75% of the IL-10delivery constructs in the first fraction are IL-10 delivery constructsin the dimer form. In some embodiments, at least 80% of the IL-10delivery constructs in the second fraction are IL-10 delivery constructsin the dimer form. In some embodiments, the method further comprisesperforming tangential flow filtration of the second fraction. In someembodiments, the tangential flow filtration comprises ultrafiltration.In some embodiments, the method further comprises diafiltration. In someembodiments, the method further comprises performing sterile filtrationthe second fraction. In some embodiments, the method does not comprisecation exchange chromatography. In some embodiments, the pool comprisesrefolded IL-10 delivery constructs.

Described herein, in certain embodiments, are oral formulationscomprising: (a) IL-10 delivery constructs; (b) one or morepharmaceutically acceptable excipients; and (c) a first coat comprisingtwo or more copolymers each having a different nominal dissolution pH;wherein the oral formulation is configured to release substantially noneof the IL-10 delivery construct after 1 h exposure to a solution havinga pH of 1.0 in a Type 4 dissolution apparatus in open mode. In someembodiments, the solution having the pH of 1.0 is a dissolution mediacontaining hydrochloric acid. In some embodiments, the oral formulationis configured to release at least 40% of the IL-10 delivery constructafter 2 hours of exposure to a solution having a pH of 7.0 in a Type 4dissolution apparatus in open mode. In some embodiments, at least 5%, atleast 10%, at least 20%, or at least 25% of the IL-10 deliveryconstructs released following 2 hours of exposure to the solution havingthe pH of 7.0 are in a dimer form. In some embodiments, the solutionhaving the pH of 7.0 is a citrate/phosphate buffer. In some embodiments,the IL-10 delivery construct comprises a carrier. In some embodiments,the carrier is derived from a polypeptide secreted by a bacterium. Insome embodiments, the bacterium is Vibrio cholerae. In some embodiments,the polypeptide secreted by Vibrio cholerae is a cholix polypeptide.

In some embodiments, the IL-10 delivery constructs have at least 80%,85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto an amino acid sequence set forth in SEQ ID NO: 5. In someembodiments, the IL-10 delivery constructs have a V1L substitution atamino acid position 1 of the carrier. In some embodiments, the oralformulation is in a capsule or a tablet. In some embodiments, a firstcopolymer has at least 50% nominal dissolution at pH >5.5 and a secondcopolymer has at least 50% nominal dissolution at pH>7.0. In someembodiments, the first copolymer comprises methacrylic acid and ethylacrylate. In some embodiments, the first polymer has a weight averagemolecular mass of from 200,000 g/mol to 450,000 g/mol, or from 250,000g/mol to 400,000 g/mol, or from 280,000 g/mol to 370,000 g/mol, or from300,000 g/mol to 340,000 g/mol. In some embodiments, the first copolymercomprises the polymer of formula I:

wherein x, y, and n are each greater than or equal to one.

In some embodiments, a ratio of free carboxyl groups to ester groups inthe first copolymer is from 0.8:1 and 1.2:1. In some embodiments, thesecond copolymer is different from the first copolymer. In someembodiments, a ratio of the first copolymer to the second copolymer inthe first coat is from 15:85 to 55:45. In some embodiments, a ratio ofthe first copolymer to the second copolymer in the first coat is 20:80,30:70, 40:60, or 50:50. In some embodiments, the second copolymercomprises methacrylic acid, methyl methacrylate, and methyl acrylate. Insome embodiments, the second polymer has a weight average molecular massof from 160,000 g/mol to 400,000 g/mol or from 200,000 g/mol to 360,000g/mol, or from 240,000 g/mol to 320,000 g/mol, or from 260,000 g/mol to300,000 g/mol. In some embodiments, wherein the second copolymercomprises the polymer of formula II:

wherein x, y, z, and n are each greater than or equal to one.

In some embodiments, a ratio of free carboxyl groups to ester groups inthe second copolymer is from 0.8:1 to 1.2:1. In some embodiments, thefirst coat further comprises an anti-tacking agent, a plasticizer, asurfactant, or a combination thereof. In some embodiments, the firstcoat comprises an anti-tacking agent, wherein the anti-tacking agentcomprises glycerol monostearate. In some embodiments, the first coatcomprises a plasticizer, wherein the plasticizer is triethyl citrate. Insome embodiments, the first coat comprises a surfactant, wherein thesurfactant is polysorbate 80. In some embodiments, from 5% to 15% (w/w)of the first coat is a mixture of glycerol monostearate, triethylcitrate, and polysorbate 80. In some embodiments, the first coat has athickness substantially equivalent to the thickness of a 60 mg coat on asize 1 capsule. In some embodiments, the first coat is disposed aroundan interior portion in an amount from 0.1 mg/mm2 to 0.2 mg/mm2. In someembodiments, the first coat has a mass from 30 mg to 60 mg. In someembodiments, the oral formulation further comprises a second coatexterior of the first coat. In some embodiments, the second coatcomprises hydroxypropyl methylcellulose (HPMC). In some embodiments, theoral formulation further comprising a third coat interior to the firstcoat and exterior of the IL-10 delivery constructs and the one or morepharmaceutically acceptable excipients. In some embodiments, the thirdcoat comprises HPMC.

In some embodiments, the IL-10 delivery constructs are present in theoral formulation in an amount from 1 mg to 20 mg. In some embodiments,the IL-10 delivery constructs are present in the oral formulation in anamount of 1 mg, 5 mg, or 20 mg. In some embodiments, the one or morepharmaceutically acceptable excipients comprise a surfactant, anosmolyte, a salt, and a bulking agent. In some embodiments, the saltcomprises potassium phosphate, the bulking agent comprises glycine, theosmolyte comprises sucrose, and the surfactant comprises poloxamer 188.In some embodiments, the oral formulation comprises a weight ratio ofthe osmolyte to the IL-10 delivery construct of from 0.45:1 to 0.55:1,preferably about 0.5:1. In some embodiments, the oral formulationcomprises a weight ratio of the surfactant to the IL-10 deliveryconstruct of from 0.12:1 to 0.18:1, preferably about 0.15:1. In someembodiments, the oral formulation comprises a weight ratio of the saltto the IL-10 delivery construct of from 0.05:1 to 0.09:1, preferablyabout 0.07:1. In some embodiments, the oral formulation comprises aweight ratio of the bulking agent to the IL-10 delivery construct offrom 0.8:1 to 1.2:1, preferably about 1:1. In some embodiments, the oralformulation is a solid. In some embodiments, the oral formulation is ina unit dose form. In some embodiments, the oral formulation has ashelf-life of at least 3 months, at least 6 months, at least 12 months,at least 18 months, or at least 24 months. In some embodiments, the oneor more pharmaceutically acceptable excipients comprise a potassiumsalt, glycine, sucrose or trehalose, and a poloxamer, wherein thepoloxamer has a weight average molecular mass of from 15,000 to 25,000daltons and a polyoxythylene content of from 70% to 90% by weight; andwherein the oral formulation further comprises: (c) a first coatcomprising a first copolymer, wherein the first copolymer comprises apolymer of formula I:

wherein x, y, and n are each greater than or equal to one; and furthercomprises a second copolymer, wherein the second copolymer comprises apolymer of formula II:

wherein x, y, z, and n are each greater than or equal to one; wherein aratio of the first copolymer to the second copolymer is 30:70; andwherein the first coat further comprises from 5% to 15% (w/w) of amixture of glycerol monostearate, triethyl citrate, and polysorbate 80;(d) a second coat comprising HPMC positioned exterior of the first coat;and (e) a third coat comprising HPMC positioned interior of the firstcoat and exterior of the therapeutic payload and the one or morepharmaceutically acceptable excipients.

Described herein, in certain embodiments, are solid compositionscomprising: IL-10 delivery constructs; and one or more excipients;wherein each of the IL-10 delivery constructs comprises IL-10 coupled toa carrier that promotes transcytosis of the IL-10 delivery constructacross a polarized gut epithelial cell; and wherein greater than 80% ofthe IL-10 delivery constructs are in a dimer form. In some embodiments,the solid composition is lyophilized or spray dried. In someembodiments, the solid composition is a tablet or a capsule. In someembodiments, the one or more pharmaceutically acceptable excipientscomprise a surfactant. In some embodiments, the surfactant is apoloxamer. In some embodiments, the poloxamer is poloxamer 188. In someembodiments, the surfactant does not include a polysorbate. In someembodiments, the one or more pharmaceutically acceptable excipientscomprise an osmolyte. In some embodiments, the osmolyte is sucrose. Insome embodiments, the one or more pharmaceutically acceptable excipientscomprise a salt. In some embodiments, the salt is potassium phosphate.In some embodiments, the one or more pharmaceutically acceptableexcipients comprise a bulking agent. In some embodiments, the bulkingagent is glycine. In some embodiments, the IL-10 has at least 80%, 85%,90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to anamino acid sequence set forth in SEQ ID NO: 2. In some embodiments, theIL-10 is coupled to the carrier via a linker. In some embodiments, thelinker comprises an amino acid sequence set forth in SEQ ID NO: 6. Insome embodiments, the carrier comprises an amino acid sequence set forthin SEQ ID NO: 4. In some embodiments, the carrier comprises an aminoacid sequence set forth in SEQ ID NO: 12. In some embodiments, the IL-10delivery construct has at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to an amino acid sequence set forthin SEQ ID NO: 5. In some embodiments, the IL-10 delivery construct has aV1L substitution at amino acid position 1 of the carrier.

In some embodiments, greater than 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, or 90% of the IL-10 delivery constructs are in a dimer form.In some embodiments, from 85% to 92% of the IL-10 delivery constructsare in a dimer form. In some embodiments, the solid compositioncomprises a first coat. In some embodiments, the first coat comprises afirst copolymer and a second copolymer, wherein the first coat isexternal of the IL-10 delivery constructs and one or more excipients. Insome embodiments, the first copolymer comprises methacrylic acid andethyl acrylate. In some embodiments, the second copolymer comprisesmethacrylic acid, methyl methacrylate, and methyl acrylate. In someembodiments, a ratio of the first copolymer to the second copolymer inthe first coat is from about 15:85 to about 55:45. In some embodiments,a ratio of the first copolymer to the second copolymer in the first coatis 20:80, 30:70, 40:60, or 50:50. In some embodiments, the solidcomposition further comprises a second coat exterior of the first coat.In some embodiments, the second coat comprises hydroxypropylmethylcellulose (HPMC). In some embodiments, the solid compositionfurther comprises a third coat interior to the first coat and exteriorof the IL-10 delivery constructs and the one or more excipients. In someembodiments, the third coat comprises HPMC.

Described herein, in certain embodiments, are solid oral formulationscomprising: (a) an IL-10 delivery construct comprising IL-10 coupled toa carrier that promotes transcytosis of IL-10 delivery construct acrossa polarized gut epithelial cell; and (b) one or more pharmaceuticallyacceptable excipients, wherein the one or more pharmaceuticallyacceptable excipients comprise a non-ionic lubricant; and (c) a firstcoat surrounding the IL-10 delivery construct and the one or morepharmaceutically acceptable excipients. In some embodiments, thenon-ionic lubricant is glyceryl behenate. In some embodiments, the oralformulation lacks magnesium stearate. In some embodiments, the oralformulation is in a tablet form. In some embodiments, the oralformulation is configured such that substantially none of the IL-10delivery construct is released from the oral formulation after 1 hexposure to a solution at pH 1.0 in a Type 4 dissolution apparatus inopen mode. In some embodiments, the oral formulation is configured torelease at least 40% of the IL-10 delivery construct after 2 hours ofexposure to a solution at pH 7.0 in a Type 4 dissolution apparatus inopen mode. In some embodiments, the oral formulation further comprises afirst coat comprising two or more copolymers each having a differentnominal dissolution pH. In some embodiments, at least 45% of the IL-10delivery construct is in a dimer form. In some embodiments, the one ormore pharmaceutically acceptable excipients comprise a bulking agent, adisintegrant, or a combination thereof. In some embodiments, the bulkingagent is silicified microcrystalline cellulose (SMCC). In someembodiments, the disintegrant is crospovidone (crosslinkedpolyvinylpyrrolidone). In some embodiments, the IL-10 delivery constructhas at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence set forth in SEQ ID NO: 5.In some embodiments, the IL-10 delivery construct has a V1L substitutionat amino acid position 1 of the carrier. In some embodiments, the oralformulation is created by compression of the IL-10 delivery constructand the one or more pharmaceutically acceptable excipients. In someembodiments, the compression occurs with a compression force of fromabout 2000 pound-force (lbf) to about 3500 lbf. In some embodiments, thefirst coat comprises a first copolymer and a second copolymer, whereinthe first coat is external of the IL-10 delivery constructs and one ormore pharmaceutically acceptable excipients. In some embodiments, thefirst copolymer comprises methacrylic acid and ethyl acrylate. In someembodiments, the second copolymer comprises methacrylic acid, methylmethacrylate, and methyl acrylate.

In some embodiments, a ratio of the first copolymer to the secondcopolymer in the first coat is from about 15:85 to about 55:45. In someembodiments, a ratio of the first copolymer to the second copolymer inthe first coat is 20:80, 30:70, 40:60, or 50:50. In some embodiments,the solid oral formulation further comprises a second coat exterior ofthe first coat. In some embodiments, the second coat compriseshydroxypropyl methylcellulose (HPMC). In some embodiments, the solidoral formulation further comprising a third coat interior to the firstcoat and exterior of the IL-10 delivery constructs and the one or morepharmaceutically acceptable excipients. In some embodiments, the thirdcoat comprises HPMC. In some embodiments, the one or morepharmaceutically acceptable excipients further comprise a potassiumsalt, glycine, sucrose or trehalose, and a poloxamer, wherein thepoloxamer has a weight average molecular mass of from 15,000 to 25,000daltons and a polyoxythylene content of from 70% to 90% by weight; andwherein the oral formulation further comprises: (c) a first coatcomprising a first copolymer, wherein the first copolymer comprises apolymer of formula

wherein x, y, and n are each greater than or equal to one; and furthercomprises a second copolymer, wherein the second copolymer comprises apolymer of formula II:

wherein x, y, z, and n are each greater than or equal to one; wherein aratio of the first copolymer to the second copolymer is 30:70; andwherein the first coat further comprises from 5% to 15% (w/w) of amixture of glycerol monostearate, triethyl citrate, and polysorbate 80;(d) a second coat comprising HPMC positioned exterior of the first coat;and (e) a third coat comprising HPMC positioned interior of the firstcoat and exterior of the therapeutic payload and the one or morepharmaceutically acceptable excipients.

Described herein, in certain embodiments, are oral formulationscomprising: (a) an IL-10 and (b) one or more pharmaceutically acceptableexcipients, wherein administration of a dose of the oral formulation toan individual results in an immunomodulatory response selected from thegroup consisting of: (i) a decrease in a concentration of fecalcalprotectin (FCP) relative to an FCP baseline, (ii) a decrease in aconcentration of C-Reactive Protein (CRP) relative to a CRP baseline,(iii) a decrease in a Geboes score relative to a Geboes score baseline,and (iv) a combination of (i)-(iii). In some embodiments, theimmunomodulatory response comprises the decrease in FCP relative to theFCP baseline. In some embodiments, the concentration of FCP isdetermined from a fecal sample or a colonic biopsy. In some embodiments,the decrease in the concentration of FCP is a decrease of at least 20%,30%, 40%, or 50% relative to the FCP baseline.

In some embodiments, the FCP baseline is an initial concentration of FCPin the individual prior to the administration. In some embodiments, theinitial concentration of FCP can be indicative of a gastrointestinalindication of the individual. In some embodiments, the initialconcentration of FCP is greater than 150 μg/g. The oral formulation ofclaim 178 or claim 179, wherein the gastrointestinal indication isulcerative colitis (UC) or Crohn's disease. In some embodiments, theconcentration of FCP is decreased at least 50% relative to the initialconcentration of FCP, and the dose of the oral formulation is from about1 mg to about 3 mg. In some embodiments, the FCP baseline is aplacebo-adjusted FCP baseline. In some embodiments, the concentration ofFCP is decreased at least 20% relative to the placebo-adjusted FCPbaseline and the dose of the oral formulation is from about 1 mg toabout 3 mg. In some embodiments, the concentration of FCP is decreasedto 50 μg/g or less. In some embodiments, the immunomodulatory responsecomprises the decrease in the concentration of CRP relative to the CRPbaseline. In some embodiments, the concentration of CRP is a systemicconcentration of CRP. In some embodiments, the concentration of CRP isdetermined from a blood sample. In some embodiments, the decrease in theconcentration of CRP is a decrease of at least 10%, 20%, 30%, 40%, 50%,60%, 70%, or 80% relative to the CRP baseline. In some embodiments, theCRP baseline is an initial concentration of CRP in the individual priorto the administration. In some embodiments, the initial concentration ofCRP is greater than 5 mg/L. In some embodiments, the initialconcentration of CRP is indicative of a gastrointestinal indication ofthe individual.

In some embodiments, the gastrointestinal indication is irritable boweldisease (IBD). In some embodiments, the concentration of CRP isdecreased at least 40% relative to the initial concentration CRP and thedose of the oral formulation is from about 1 mg to about 3 mg. In someembodiments, the CRP baseline is a placebo-adjusted CRP baseline. Insome embodiments, the concentration of CRP is decreased at least 10%relative to the placebo-adjusted CRP baseline and the dose of the oralformulation is about 3 mg. In some embodiments, the concentration of CRPis decreased at least 40% relative to placebo-adjusted CRP baseline andthe dose of the oral formulation is about 1 mg. In some embodiments, theconcentration of CRP is decreased to less than 5 mg/L. In someembodiments, the immunomodulatory response comprises the decrease in theGeboes score relative to the Geboes score baseline. In some embodiments,the Geboes score baseline is an initial Geboes score prior to theadministration. In some embodiments, the Geboes score baseline is aplacebo-adjusted Geboes score baseline. In some embodiments, the Geboesscore is decreased a least 2 units relative to the placebo-adjustedGeboes score baseline and the dose of the oral formulation is from about1 mg to about 30 mg.

In some embodiments, less than 5% of the administered IL-10 enters thebloodstream of the individual. In some embodiments, the immunomodulatoryresponse is observed after daily administration of the dose of the oralformulation for 14 days. In some embodiments, the dose of the oralformulation is 10 mg or less. In some embodiments, the dose of the oralformulation is from 1 mg to 10 mg, from 3 mg to 10 mg, or from 1 mg to 3mg. In some embodiments, the dose of the oral formulation is 1 mg, 3 mg,or 10 mg. In some embodiments, the oral formulation is a capsule.

In some embodiments, the oral formulation is biodegradable. In someembodiments, the one or more pharmaceutically acceptable excipientscomprise a surfactant. In some embodiments, the surfactant is poloxamer188. In some embodiments, the one or more pharmaceutically acceptableexcipients comprise an osmolyte. In some embodiments, the osmolyte issucrose. In some embodiments, the one or more pharmaceuticallyacceptable excipients comprise a salt. In some embodiments, the salt ispotassium phosphate. In some embodiments, the one or morepharmaceutically acceptable excipients comprise a bulking agent. In someembodiments, the bulking agent is glycine. In some embodiments, the oralformulation comprises a first coat, wherein the first coat is externalof the IL-10 and one or more pharmaceutically acceptable excipients. Insome embodiments, the first coat comprises a first copolymer comprisingmethacrylic acid and ethyl acrylate and a second copolymer comprisingmethacrylic acid, methyl methacrylate, and methyl acrylate. In someembodiments, a ratio of the first copolymer to the second copolymer inthe first coat is from about 15:85 to 55:45. In some embodiments, aratio of the first copolymer to the second copolymer in the first coatis 20:80, 30:70, 40:60, or 50:50.

In some embodiments, the oral formulation further comprises a secondcoat located interior of the first coat and external of the IL-10 andone or more pharmaceutically acceptable excipients. In some embodiments,the second coat comprises hydroxypropyl methylcellulose (HPMC). In someembodiments, the oral formulation further comprises a third coatinterior to the first coat and exterior of the IL-10 and the one or morepharmaceutically acceptable excipients. In some embodiments, the thirdcoat comprises HPMC. In some embodiments, the IL-10 is part of an IL-10delivery construct having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to an amino acid sequence setforth in SEQ ID NO: 5. In some embodiments, the IL-10 delivery constructcomprises a carrier. In some embodiments, the IL-10 delivery constructhas a V1L substitution at amino acid position 1 of the carrier. In someembodiments, the one or more pharmaceutically acceptable excipientscomprise a potassium salt, glycine, sucrose or trehalose, and apoloxamer, wherein the poloxamer has a weight average molecular mass offrom 15,000 to 25,000 daltons and a polyoxythylene content of from 70%to 90% by weight; and wherein the oral formulation further comprises:(c) a first coat comprising a first copolymer, wherein the firstcopolymer comprises a polymer of formula

wherein x, y, and n are each greater than or equal to one; and furthercomprises a second copolymer, wherein the second copolymer comprises apolymer of formula II:

wherein x, y, z, and n are each greater than or equal to one; wherein aratio of the first copolymer to the second copolymer is 30:70; andwherein the first coat further comprises from 5% to 15% (w/w) of amixture of glycerol monostearate, triethyl citrate, and polysorbate 80;(d) a second coat comprising HPMC positioned exterior of the first coat;and (e) a third coat comprising HPMC positioned interior of the firstcoat and exterior of the therapeutic payload and the one or morepharmaceutically acceptable excipients.

Described herein, in certain embodiments, are methods of treating aninflammatory disorder in an individual, the method comprisingadministering to the individual an oral formulation comprising IL-10 andone or more pharmaceutically acceptable excipients to the individual,wherein the administering results in an immunomodulatory responseselected from the group consisting of: (i) a decrease in a concentrationof fecal calprotectin (FCP) relative to an FCP baseline, (ii) a decreasein a concentration of C-Reactive Protein (CRP) relative to a CRPbaseline, (iii) a decrease in a Geboes score relative to a baselineGeboes score, and (iv) a combination of (i)-(iii). In some embodiments,the inflammatory disorder is selected from the group consisting ofulcerative colitis, inflammatory bowel disease (IBD), proctitis,pouchitis, Crohn's disease, Celiac disease, multiple sclerosis (MS),systemic lupus erythematosus (SLE), graft versus host disease (GVHD),rheumatoid arthritis, psoriatic arthritis, or psoriasis.

Described herein, in certain embodiments, are methods of modulating abiomarker in an individual with an inflammatory disorder, the methodcomprising administering to the individual an oral formulationcomprising IL-10 and one or more pharmaceutically acceptable excipientsto the individual, wherein the biomarker is selected from the groupconsisting of: fecal calprotectin (FCP), C-Reactive Protein (CRP), and acombination thereof. In some embodiments, the administering results in adecrease in a concentration of the FCP relative to an FCP baseline. Insome embodiments, the administering results in a decrease in aconcentration of the CRP relative to a CRP baseline. In someembodiments, the administering further results in a decrease in a Geboesscore relative to a baseline Geboes score. In some embodiments,comprising treating the individual with the inflammatory disorder. Insome embodiments, the inflammatory disorder is selected from the groupconsisting of ulcerative colitis, inflammatory bowel disease (IBD),proctitis, pouchitis, Crohn's disease, Celiac disease, multiplesclerosis (MS), systemic lupus erythematosus (SLE), graft versus hostdisease (GVHD), rheumatoid arthritis, psoriatic arthritis, or psoriasis.

Described herein, in certain embodiments, are methods of treating aninflammatory disorder in an individual refractory or resistant to atleast one anti-inflammatory agent, the method comprising administering aformulation comprising IL-10 to the individual. In some embodiments, theanti-inflammatory agent is an aminosalicylate. In some embodiments, theaminosalicylate is selected from the group consisting of5-aminosalicylic acid (5-ASA; mesalazine), 4-amino salacylic acid(4-ASA), balsalazide, olsalazine, and sulfasalazine. In someembodiments, the anti-inflammatory agent is a corticosteroid. In someembodiments, the corticosteroid is prednisone. In some embodiments, thecorticosteroid is an orally administered corticosteroid or anintravenously (IV) administered corticosteroid. In some embodiments, theanti-inflammatory agent is an immunosuppressive agent. In someembodiments, the immunosuppressive agent is selected from the groupconsisting of azathioprine, 6-mercaptopurine, and a combination thereof.In some embodiments, the anti-inflammatory agent is a TNFα inhibitor. Insome embodiments, the TNFα inhibitor is selected from the groupconsisting of adalimumab, certolizumab, etanercept, golimumab, andinfliximab.

In some embodiments, the at least one anti-inflammatory agent is a Januskinase (JAK) inhibitor. In some embodiments, the JAK inhibitor isselected from the group consisting of filgotinib, upadacitinib,peficitinib, and tofacitinib. In some embodiments, the at least oneanti-inflammatory agent is a sphingosine-1-phosphate (SIP) receptorantagonist. In some embodiments, the SIP receptor antagonist is selectedfrom the group consisting of ozanimod, amiselimod, and etrasimod. Insome embodiments, the at least one anti-inflammatory agent is anintegrin blocker. In some embodiments, the integrin blocker is selectedfrom the group consisting of etrolizumab, natalizumab, vedolizumab,abrilumab, and carotegrast methyl. In some embodiments, the at least oneanti-inflammatory agent is an IL-23 inhibitor. In some embodiments, theIL-23 inhibitor is selected from the group consisting of ustekinumab.mirikizumab, brazikumab, guselkumab, and risankizumab. In someembodiments, the at least one anti-inflammatory agent is aphosphodiesterase 4 (PDE4) inhibitor. In some embodiments, the at leastone PDE4 inhibitor is selected from the group consisting of apremilast,cilomilast, roflumilast, tetomilast, and rolipram. In some embodiments,the at least one anti-inflammatory agent is laquinimod.

Described herein, in certain embodiments, are methods of refolding anIL-10 delivery construct, the method comprising: (i) contactinginclusion bodies comprising the IL-10 delivery construct with asolubilization solution comprising a chaotropic agent to produce asoluble IL-10 delivery construct; (ii) contacting the soluble IL-10delivery construct with a sulfitolysis reducing agent to produce areduced IL-10 delivery construct; and (iii) contacting the reduced IL-10delivery construct with a refolding solution comprising reducedglutathione and oxidized glutathione to produce a refolded IL-10delivery construct. Described herein, in certain embodiments, aremethods of refolding an IL-10 delivery construct, the method comprising:(i) contacting a soluble IL-10 delivery construct with a sulfitolysisreducing agent comprising sodium sulfite to produce a reduced IL-10delivery construct; (ii) contacting the reduced IL-10 delivery constructwith potassium tetrathionate; (iii) clarifying the reduced IL-10delivery construct by depth filtration to produce a clarified IL-10delivery construct; (iv) performing ultrafiltration followed bydiafiltration on the clarified IL-10 delivery construct; and (v)contacting the clarified IL-10 delivery construct with a refoldingsolution comprising: from 0.8 mM to 1.2 mM of reduced glutathione, from0.4 mM to 0.6 mM of oxidized glutathione, from 800 mM to 1.2M arginine,from 200 mM to 300 mM sucrose, from 75 mM to 125 mM Tris, and from 1.5mM to 2.5 mM EDTA, wherein the refolding solution is buffered at a pHfrom 7.5 to 8.5 to produce a refolded IL-10 delivery construct, andwherein the contacting with the refolding solution occurs for at least16 hours. In some embodiments, the sulfitolysis reducing agent comprisessodium sulfite.

Described herein, in certain embodiments, are oral formulationscomprising IL-10 and one or more pharmaceutically acceptable excipients,wherein administration of a dose of the oral formulation from about 1 mgto about 60 mg to an individual results in a greater than 20% increasein a plasma concentration of IL-1Ra in the individual relative to abaseline plasma concentration of IL-1Ra. Described herein, in certainembodiments, are methods of treating an inflammatory disorder in anindividual comprising administering a dose of an oral formulationcomprising IL-10 and one or more pharmaceutically acceptable excipientsto the individual, wherein the administering results in a greater than20% increase in a plasma concentration of IL-1Ra in the individualrelative to a baseline plasma concentration of IL-1Ra. In someembodiments, the dose of the oral formulation is from about 3 mg toabout 30 mg and the increase in the plasma concentration of IL-1Rarelative to the baseline plasma concentration of IL-1Ra is greater than30%. In some embodiments, the dose of the oral formulation is from about3 mg to about 30 mg and the increase in the plasma concentration ofIL-1Ra relative to the baseline plasma concentration of IL-1Ra is from30% to 45%, 30% to 35%, or from 40% to 43%. In some embodiments,administration of the dose of the oral formulation to the individualresults in a plasma concentration of IL-10 in the individual that doesnot exceed 1500 pg/mL, 1000 pg/mL, 500 pg/mL, 100 pg/mL, or 10 pg/mL. Insome embodiments, administration of the oral formulation to theindividual results in co-localization of the IL-10 with a cellexpressing CD3 in a lamina propria of the individual. In someembodiments, the cell expressing CD3 is a lymphocyte. In someembodiments, the lymphocyte is a T lymphocyte. In some embodiments,administration of the oral formulation to the individual results inco-localization of the IL-10 with a macrophage in the lamina propria ofthe individual. In some embodiments, administration of the oralformulation to the individual does not result in co-localization of theIL-10 with a cell in the lamina propria of the individual, wherein thecell is selected from the group consisting of a dendritic cell, aB-lymphocyte, an endothelial cell, and a combination thereof.

Described herein, in certain embodiments, are oral formulationscomprising IL-10 and one or more pharmaceutically acceptable excipients,wherein administration of the oral formulation to an individual resultsin an increase in a concentration of IL-1Ra in plasma of the individualof at least 5000 pg/mL relative to baseline levels and at least one ofthe following: (1) a peak IL-10 plasma concentration of less than 50pg/mL and (2) co-localization of the IL-10 with a cell expressing CD3 ina lamina propria of the individual. Described herein, in certainembodiments, are methods of treating an inflammatory disorder in anindividual comprising administering an oral formulation comprising IL-10and one or more pharmaceutically acceptable excipients to theindividual, wherein the administering results in an increase in aconcentration of IL-1Ra in plasma of the individual of at least 5000pg/mL relative to baseline levels and at least one of the following (1)a peak IL-10 plasma concentration of less than 50 pg/mL and (2)co-localization of the IL-10 with a cell expressing CD3 in a laminapropria of the individual. In some embodiments, the inflammatorydisorder is selected from the group consisting of ulcerative colitis,proctitis, pouchitis, Crohn's disease, multiple sclerosis (MS), systemiclupus erythematosus (SLE), graft versus host disease (GVHD), rheumatoidarthritis, or psoriasis. In some embodiments, the peak IL-1Raconcentration in plasma of the individual is obtained from 2 to 4 hours,or from 2 to 3 hours, after the administration. In some embodiments,administration of the oral formulation to the individual results in apeak IL-10 concentration in plasma of the individual of less than 10pg/mL, 2.5 pg/mL, or 1.5 pg/mL. In some embodiments, the concentrationof IL-1Ra reaches a maximum of from 25,000 pg/mL to 28,000 pg/mL. Insome embodiments, administration of the oral formulation to anindividual results in an increase in a ratio of expression of IL-Ra tointerleukin 1 beta in the colonic tissue of the individual. In someembodiments, the ratio of IL-1Ra to IL-1 beta is at least 2:1. In someembodiments, administration of the oral formulation to an individualresults in an increase in expression of interleukin 1 receptor agonist(IL-1Ra) in a colonic tissue of the individual.

Described herein, in certain embodiments, are non-natural nucleic acidscomprising a sequence that has at least 90%, at least 92%, at least 95%,at least 98%, or at least 99% sequence identity to SEQ ID NO: 10. Insome embodiments, the non-natural nucleic acid has a sequence with 100%identity to SEQ ID NO: 10.

Described herein, in certain embodiments, are solid oral formulationscomprising (i) an IL-10 delivery construct comprising IL-10 coupled to acarrier that promotes transcytosis of the IL-10 delivery constructacross a polarized gut epithelial cell; (ii) one or more excipients,wherein the one or more excipients comprise a non-ionic lubricant; and(iii) a first coat surrounding the IL-10 delivery construct and the oneor more excipients. In some embodiments, the non-ionic lubricant isglyceryl behenate. In some embodiments, the oral formulation lacks anionic surfactant. In some embodiments, the oral formulation lacksmagnesium stearate. In some embodiments, the oral formulation is in atablet form.

In some embodiments, the oral formulation is configured such thatsubstantially none of the IL-10 delivery construct is released from theoral formulation after 1 h exposure to a solution at pH 1.0 in a Type 4dissolution apparatus in open mode. In some embodiments, the oralformulation is configured to release at least 40% of the IL-10 deliveryconstruct after 2 hours of exposure to a solution at pH 7.0 in a Type 4dissolution apparatus in open mode. In some embodiments, the first coatcomprises a blend of polymers each having a different nominaldissolution pH. In some embodiments, at least 45% of the IL-10 deliveryconstruct is in a dimer form.

In some embodiments, the one or more excipients comprise a bulkingagent, a disintegrant, or a combination thereof. In some embodiments,the bulking agent is silicified microcrystalline cellulose (SMCC). Insome embodiments, the disintegrant is crospovidone (crosslinkedpolyvinylpyrrolidone). In some embodiments, the IL-10 delivery constructhas at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence set forth in SEQ ID NO: 5.In some embodiments, the IL-10 delivery construct has at least 80%, 85%,90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to anamino acid sequence set forth in SEQ ID NO: 13.

Described herein, in certain embodiments, are oral formulations intablet form comprising IL-10 and one or more pharmaceutically acceptableexcipients encapsulated by an enteric coating, wherein, following 1 hrof submersion of the oral formulation into a solution at pH 7.0 in aType 4 dissolution apparatus, a percentage of IL-10 in dimer form is atleast 45%. In some embodiments, the solution at pH 7.0 is acitrate/phosphate buffer. In some embodiments, the enteric coating has athickness of from 4 mg/cm² to 20 mg/cm², from 4 mg/cm² to 6 mg/cm², from5 mg/cm² to 10 mg/cm², or from 5 mg/cm² to 20 mg/cm². In someembodiments, the IL-10 comprises at least 80%, 85%, 90%, 92%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequenceset forth in SEQ ID NO: 2.

In some embodiments, the one or more pharmaceutically acceptableexcipients comprise a surfactant. In some embodiments, the surfactant ispoloxamer 188. In some embodiments, the one or more pharmaceuticallyacceptable excipients comprise an osmolyte. In some embodiments, theosmolyte is sucrose. In some embodiments, the one or morepharmaceutically acceptable excipients comprise a salt. In someembodiments, the salt is potassium phosphate. In some embodiments, theone or more pharmaceutically acceptable excipients comprise a bulkingagent. In some embodiments, the bulking agent is glycine. In someembodiments, the one or more pharmaceutically acceptable excipientscomprises at least one compacting excipient. In some embodiments, the atleast one compacting excipient comprises a bulking agent. In someembodiments, the bulking agent is silicified microcrystalline cellulose(SMCC). In some embodiments, the at least one compacting excipientcomprises a disintegrant. In some embodiments, the disintegrant iscrospovidone (crosslinked polyvinylpyrrolidone). In some embodiments,the at least one compacting excipient comprises a lubricant. In someembodiments, the lubricant is a non-ionic surfactant. In someembodiments, the non-ionic surfactant is glyceryl behenate. In someembodiments, the non-ionic surfactant is glyceryl dibehenate. In someembodiments, the at least one compacting excipient is comprised in anintragranular phase, an extragranular phase, or a combination thereof.

In some embodiments, the oral formulation is created by compression ofthe IL-10 and the at least one compacting excipients. In someembodiments, the compression occurs with a compression force of fromabout 2000 pound-force (lbf) to about 3500 lbf.

In some embodiments, the IL-10 is part of an IL-10 delivery constructhaving at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the IL-10 is part of an IL-10 delivery constructhaving at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to an amino acid sequence set forth in SEQ ID NO:13.

In some embodiments, the enteric coating comprises a first copolymer anda second copolymer, wherein the enteric coating is external of the IL-10and one or more pharmaceutically acceptable excipients. In someembodiments, the first copolymer comprises methacrylic acid and ethylacrylate. In some embodiments, the second copolymer comprisesmethacrylic acid, methyl methacrylate, and methyl acrylate. In someembodiments, a ratio of the first copolymer to the second copolymer inthe enteric coating is from about 50:50 to about 20:80 by weight. Insome embodiments, the ratio of the first copolymer to the secondcopolymer in the enteric coating is from about 25:75 to about 35:65 byweight. In some embodiments, the enteric coating is from 5% to 12% ofthe weight of the oral formulation. In some embodiments, the entericcoating is no more than 12% of the weight of the oral formulation. Insome embodiments, the oral formulation further comprises a secondenteric coating located interior of the enteric coating and external ofthe IL-10 and one or more pharmaceutically acceptable excipients. Insome embodiments, the second enteric coating comprises hydroxypropylmethylcellulose (HPMC). In some embodiments, the second enteric is from3% to 5% of the weight of the oral formulation. In some embodiments, thepercentage of IL-10 in dimer form is at least 45% when the oralformulation is in a solid form.

In some embodiments, the enteric coating comprises hydroxypropylmethylcellulose acetate succinate (HPMCAS), wherein the enteric coatingis external of the IL-10 and one or more pharmaceutically acceptableexcipients. In some embodiments, the HPMCAS comprises a first HPMCAS anda second HPMCAS. In some embodiments, the first HPMCAS is soluble at apH of greater than or equal to 6.8. In some embodiments, the firstHPMCAS comprises HPMCAS-HF. In some embodiments, the second HPMCAS issoluble at a pH of greater than or equal to 6.0. In some embodiments,the second HPMCAS comprises HPMCAS-MF. In some embodiments, a ratio ofthe first HPMCAS to the second HPMCAS is from about 40:60 to about60:40.

Disclosed herein, in certain embodiments, are methods comprisingadministering any of the formulations described herein for treatment ofa disease or condition in an individual in need thereof. Similarly,disclosed herein are the IL-10 delivery constructs or formulationsdescribed herein for use in treating a disease or condition in anindividual in need thereof. Similarly, disclosed herein is the use of anIL-10 delivery construct as disclosed herein in the manufacture of amedicament for treating a disease or condition in an individual in needthereof. In some embodiments, the disease or condition is selected fromthe group consisting of ulcerative colitis, inflammatory bowel disease(IBD), Celiac disease, proctitis, pouchitis, Crohn's disease, multiplesclerosis (MS), systemic lupus erythematosus (SLE), graft versus hostdisease (GVHD), rheumatoid arthritis, psoriatic arthritis, andpsoriasis.

Disclosed herein, in certain embodiments, are solid compositionscomprising: IL-10 delivery constructs; and one or more excipients;wherein each of the IL-10 delivery constructs comprises IL-10 coupled toa carrier that promotes transcytosis of the IL-10 delivery constructacross a polarized gut epithelial cell; and wherein greater than 80% ofthe IL-10 delivery constructs are in a dimer form. In some embodiments,the carrier comprises an amino acid sequence set forth in SEQ ID NO: 4.In some embodiments, the carrier comprises an amino acid sequence setforth in SEQ ID NO: 12. In some embodiments, the carrier comprises anamino acid sequence comprising positions 1-386 of any one of SEQ ID NOS:20-146. In some embodiments, the carrier comprises an amino acidsequence comprising positions 1-386 of SEQ ID NO: 147.

In some embodiments, the solid composition is a tablet or a capsule. Insome embodiments, the one or more excipients comprise poloxamer 188,sucrose, potassium phosphate, glycine, or a combination thereof. In someembodiments, the IL-10 comprises at least 80%, 85%, 90%, 92%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequenceset forth in SEQ ID NO: 2. In some embodiments, the IL-10 is coupled tothe carrier via a linker. In some embodiments, the linker comprises anamino acid sequence set forth in SEQ ID NO: 6. In some embodiments,greater than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of theIL-10 delivery constructs are in a dimer form. In some embodiments, from85% to 92% of the IL-10 delivery constructs are in a dimer form.

In some embodiments, the solid composition comprises an enteric coating.In some embodiments, the enteric coating comprises a first copolymer anda second copolymer, wherein the enteric coating is external of the IL-10delivery construct and one or more excipients. In some embodiments, thefirst copolymer comprises methacrylic acid and ethyl acrylate. In someembodiments, the second copolymer comprises methacrylic acid, methylmethacrylate, and methyl acrylate. In some embodiments, a ratio of thefirst copolymer to the second copolymer in the enteric coating is fromabout 50:50 to about 20:80. In some embodiments, a ratio of the firstcopolymer to the second copolymer in the enteric coating is from about25:75 to about 35:65.

Disclosed herein, in certain embodiments, are methods of refolding anIL-10 delivery construct, the method comprising: (i) contactinginclusion bodies comprising the IL-10 delivery construct with asolubilization solution comprising a chaotropic agent to produce asoluble IL-10 delivery construct; (ii) contacting the soluble IL-10delivery construct with a refolding solution comprising reducedglutathione and oxidized glutathione to produce a refolded IL-10delivery construct; wherein the method does not comprise contacting thesoluble IL-10 delivery construct with a sulfitolysis agent or a reducingagent prior to the contacting of step (ii). In some embodiments, theIL-10 delivery construct comprises IL-10 coupled to a carrier. In someembodiments, the carrier comprises an amino acid sequence set forth inSEQ ID NO: 4. In some embodiments, the carrier comprises an amino acidsequence set forth in SEQ ID NO: 12. In some embodiments, the carriercomprises an amino acid sequence comprising positions 1-386 of any oneof SEQ ID NOS: 20-146. In some embodiments, the carrier comprises anamino acid sequence comprising positions 1-386 of SEQ ID NO: 147.

In some embodiments, the refolding solution comprises a ratio (w/w) ofthe reduced glutathione to the oxidized glutathione from 0.8:1 to 1.2:1.In some embodiments, the refolding solution comprises from 0.75 mM to1.5 mM reduced glutathione. In some embodiments, the refolding solutioncomprises from 0.25 mM to 0.75 mM oxidized glutathione. In someembodiments, the refolding solution comprises a pH from 7.5 to 8.5. Insome embodiments, the refolding solution comprises arginine, Tris, andEDTA. In some embodiments, the refolding solution comprises sucrose. Insome embodiments, the refolding solution comprises arginine, sucrose,Tris, EDTA, or a combination thereof. In some embodiments, arginine ispresent in the refolding solution at a concentration of between 900 mMand 1.1 M. In some embodiments, sucrose is present in the refoldingsolution at a concentration of between 200 mM and 300 mM. In someembodiments, Tris is present in the refolding solution at aconcentration of from 75 mM to 125 mM. In some embodiments, EDTA ispresent in the refolding solution at a concentration of from 1.75 mM to2.25 mM.

In some embodiments, the method further comprises lysing a cellcomprising the inclusion bodies. In some embodiments, the cell is abacterium. In some embodiments, the bacterium is Escherichia coli. Insome embodiments, the lysing comprises high-pressure homogenization. Insome embodiments, the method further comprises isolating the inclusionbodies. In some embodiments, the chaotropic agent comprises guanidinehydrochloride or urea. In some embodiments, the solubilization solutionfurther comprises Tris.

In some embodiments, the contacting the soluble IL-10 delivery constructwith a refolding solution occurs for at least 16 hours. In someembodiments, the contacting the soluble IL-10 delivery construct with arefolding solution occurs from 12 hours to 18 hours. In someembodiments, the contacting the IL-10 delivery construct with arefolding solution occurs from 2° C. to 8° C. In some embodiments, themethod further comprises a first sterile filtration of the refoldedIL-10 delivery construct. In some embodiments, the first sterilefiltration occurs after the contacting with the refolding solution. Insome embodiments, the method further comprises performing a tangentialflow filtration of the refolded IL-10 delivery construct. In someembodiments, the tangential flow filtration comprises diafiltration. Insome embodiments, the diafiltration comprises a first diavolume, asecond diavolume, a third diavolume, and a fourth diavolume. In someembodiments, the first diavolume and the second diavolume comprise acold buffer. In some embodiments, the third diavolume and the fourthdiavolume comprise a room temperature buffer. In some embodiments, thecold buffer and the room temperature buffer comprise Tris and NaCl.

In some embodiments, IL-10 delivery construct dimers may be stored inbuffer, for example at 25° C. for two days. Such a buffer may comprise asalt such as 1×PBS, 150 mM, or 200 mM NaCl buffered in 10 mM SodiumPhosphate at pH 7.0. IL-10 delivery construct dimers may be more stablewhen stored in a buffer comprising a salt such as 1×PBS, 150 mM, or 200mM NaCl buffered in 10 mM Sodium Phosphate at pH 7.0 than in a buffercomprising 10 mM Sodium Phosphate at pH 7.0 alone.

Described herein, in certain embodiments, are methods of enriching forIL-10 delivery construct dimers from a pool comprising IL-10 deliveryconstructs in a dimer form, a monomer form, and an aggregate form, themethod comprising: (i) performing anion exchange (AEX) chromatography onthe pool by binding the IL-10 delivery construct dimers to an anionexchange column and subsequently eluting the IL-10 delivery constructdimers from the anion exchange column, thereby creating a firstplurality of fractions, one of which is a first fraction enriched inIL-10 delivery constructs in the dimer form; and (ii) performing ceramichydroxyapatite (CHT) chromatography on the fraction enriched in IL-10delivery constructs in the dimer form, thereby creating a secondplurality of fractions, one of which is a second fraction furtherenriched in IL-10 delivery constructs in the dimer form. In someembodiments, each of the IL-10 delivery constructs comprises IL-10coupled to a carrier. In some embodiments, the carrier comprises anamino acid sequence set forth in SEQ ID NO: 4. In some embodiments, thecarrier comprises an amino acid sequence set forth in SEQ ID NO: 12. Insome embodiments, the carrier comprises an amino acid sequencecomprising positions 1-386 of any one of SEQ ID NOS: 20-146. In someembodiments, the carrier comprises an amino acid sequence comprisingpositions 1-386 of SEQ ID NO: 147.

In some cases, the method can comprise performing cation exchangechromatography, for example with a Sulfate 650F column. The cationexchange chromatography step may be performed after an anion exchangechromatography step and a ceramic hydroxyapatite (CHT) purificationstep, before an anion exchange chromatography step and a ceramichydroxyapatite (CHT) purification step, or between an anion exchangechromatography step and a ceramic hydroxyapatite (CHT) purificationstep.

In some embodiments, the method further comprises determining thepercentage of IL-10 delivery constructs in the dimer form in eachfraction of the first plurality of fractions. In some embodiments, thedetermining is by size exclusion chromatography. In some embodiments,the size exclusion chromatography is size exclusion high performanceliquid chromatography (SE-HPLC). In some embodiments, the method furthercomprises determining the percentage of IL-10 delivery constructs in thedimer form in each fraction of the second plurality of fractions. Insome embodiments, the determining is by size exclusion chromatography.In some embodiments, the size exclusion chromatography is size exclusionhigh performance liquid chromatography (SE-HPLC).

In some embodiments, at least 75% of the IL-10 delivery constructs inthe first fraction are IL-10 delivery constructs in the dimer form. Insome embodiments, at least 80% of the IL-10 delivery constructs in thesecond fraction are IL-10 delivery constructs in the dimer form. In someembodiments, the method further comprises performing tangential flowfiltration of the second fraction. In some embodiments, the tangentialflow filtration comprises ultrafiltration. In some embodiments, themethod further comprises diafiltration. In some embodiments, the methodfurther comprises performing sterile filtration the second fraction. Insome embodiments, the method does not comprise cation exchangechromatography. In some embodiments, the pool comprises refolded IL-10delivery constructs obtained from any of the methods described herein.

Described herein, in certain embodiments, are methods of treating aninflammatory disorder in an individual refractory or resistant to atleast one anti-inflammatory agent, the method comprising administering aformulation comprising IL-10 to the individual. Similarly, disclosedherein are the IL-10 delivery constructs or formulations describedherein for use in treating an inflammatory disorder in an individualrefractory or resistant to at least one anti-inflammatory agent.Similarly, disclosed herein are the use of an IL-10 delivery constructas disclosed herein in the manufacture of a medicament for treating aninflammatory disorder in an individual refractory or resistant to atleast one anti-inflammatory agent. In some embodiments, theanti-inflammatory agent is an aminosalicylate. In some embodiments, theaminosalicylate is selected from the group consisting of5-aminosalicylic acid (5-ASA; mesalazine), 4-amino salicylic acid(4-ASA), balsalazide, olsalazine, and sulfasalazine. In someembodiments, the anti-inflammatory agent is a corticosteroid. In someembodiments, the corticosteroid is prednisone. In some embodiments, thecorticosteroid is an orally administered corticosteroid or anintravenously (IV) administered corticosteroid. In some embodiments, theanti-inflammatory agent is an immunosuppressive agent. In someembodiments, the immunosuppressive agent is selected from the groupconsisting of azathioprine, 6-mercaptopurine, and a combination thereof.In some embodiments, the anti-inflammatory agent is a TNFα inhibitor. Insome embodiments, the TNFα inhibitor is selected from the groupconsisting of adalimumab, certolizumab, etanercept, golimumab, andinfliximab. In some embodiments, the at least one anti-inflammatoryagent is a Janus kinase (JAK) inhibitor. In some embodiments, the JAKinhibitor is selected from the group consisting of filgotinib,upadacitinib, peficitinib, and tofacitinib. In some embodiments, the atleast one anti-inflammatory agent is a sphingosine-1-phosphate (SIP)receptor antagonist. In some embodiments, the SIP receptor antagonist isselected from the group consisting of ozanimod, amiselimod, andetrasimod. In some embodiments, the at least one anti-inflammatory agentis an integrin blocker. In some embodiments, the integrin blocker isselected from the group consisting of etrolizumab, natalizumab,vedolizumab, abrilumab, and carotegrast methyl. In some embodiments, theat least one anti-inflammatory agent is an IL-23 inhibitor. In someembodiments, the IL-23 inhibitor is selected from the group consistingof ustekinumab. mirikizumab, brazikumab, guselkumab, and risankizumab.In some embodiments, the at least one anti-inflammatory agent is aphosphodiesterase 4 (PDE4) inhibitor. In some embodiments, the at leastone PDE4 inhibitor is selected from the group consisting of apremilast,cilomilast, roflumilast, tetomilast, and rolipram. In some embodiments,the at least one anti-inflammatory agent is laquinimod. In someembodiments, the individual is administered the formulation daily for atleast 5, 7, 10, 12, or 14 days.

In some embodiments, the inflammatory disorder is selected from thegroup consisting of ulcerative colitis, proctitis, pouchitis, Crohn'sdisease, multiple sclerosis (MS), systemic lupus erythematosus (SLE),graft versus host disease (GVHD), rheumatoid arthritis, or psoriasis. Insome embodiments, the IL-10 comprises at least 80%, 85%, 90%, 92%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acidsequence set forth in SEQ ID NO: 2. In some embodiments, the one or morepharmaceutically acceptable excipients comprise a surfactant. In someembodiments, the surfactant is selected from the group consisting of:polysorbate 80, polysorbate 20, and poloxamer 188. In some embodiments,the one or more pharmaceutically acceptable excipients comprise anosmolyte. In some embodiments, the osmolyte is selected from the groupconsisting of sucrose and trehalose. In some embodiments, the one ormore pharmaceutically acceptable excipients comprise a salt. In someembodiments, the salt is selected from the group consisting of potassiumphosphate, sodium chloride, potassium chloride, magnesium chloride, andsodium sulfate. In some embodiments, the one or more pharmaceuticallyacceptable excipients comprise a bulking agent. In some embodiments, thebulking agent is selected from the group consisting of: glycine andmannitol. In some embodiments, the one or more pharmaceuticallyacceptable excipients comprise a disintegrant. In some embodiments, thedisintegrant is selected from the group consisting of: microcrystallinecellulose (MCC), silicified microcrystalline cellulose (SMCC), starch,sodium starch glycolate, veegum, bentonite, alginic acid, calciumalginate, croscarmellose sodium (crosslinked sodium carboxymethylcellulose), and crospovidone (crosslinked polyvinylpyrrolidone). In someembodiments, the one or more pharmaceutically acceptable excipientscomprise a binding agent. In some embodiments, the binding agent isselected from the group consisting of: sucrose, lactose, starch,cellulose, gelatin, polyvinylpyrrolidone (PVP), and polyethylene glycol(PEG). In some embodiments, the one or more pharmaceutically acceptableexcipients comprise a lubricant. In some embodiments, the lubricant isselected from the group consisting of: magnesium stearate, glycerylbehenate, glyceryl dibehenate, sodium stearyl fumerate, stearic acid,talc, silica, calcium stearate, magnesium carbonate, hydrogenated oil,mineral oil, polyethylene glycol (PEG), and glyceryl monostearate.

In some embodiments, the IL-10 is part of an IL-10 delivery constructcomprising the IL-10 coupled to a carrier. In some embodiments, theIL-10 delivery construct comprises a linker coupling the IL-10 to thecarrier. In some embodiments, the linker comprises an amino acidsequence set forth in SEQ ID NO: 6. In some embodiments, the carrier hasat least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence set forth in SEQ ID NO: 4.In some embodiments, the IL-10 delivery construct has at least 80%, 85%,90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to anamino acid sequence set forth in SEQ ID NO: 5. In some embodiments, uponcontact with a cell, the carrier promotes endocytosis or transcytosis ofthe IL-10 delivery construct. In some embodiments, the cell is a gutepithelial cell. In some embodiments, the gut epithelial cell is apolarized gut epithelial cell.

In some embodiments, the formulation is an oral formulation. In someembodiments, the oral formulation is a capsule or a tablet. In someembodiments, the oral formulation comprises a first coating comprising afirst copolymer and a second copolymer, wherein the first coating isexternal of the IL-10 and one or more pharmaceutically acceptableexcipients. In some embodiments, the first copolymer comprisesmethacrylic acid and ethyl acrylate. In some embodiments, the secondcopolymer comprises methacrylic acid, methyl methacrylate, and methylacrylate. In some embodiments, a ratio (w/w) of the first copolymer tothe second copolymer in the first coat is from 0.8:1 to 1.2:1. In someembodiments, the administering comprises oral administration.

Disclosed herein, in certain embodiments, are methods of preventing arecurrence of an inflammatory disorder in an individual in remission forthe inflammatory disorder comprising administering a formulationcomprising IL-10 and one or more pharmaceutically acceptable excipientsto the individual. In some embodiments, the inflammatory disorder isselected from the group consisting of ulcerative colitis, proctitis,pouchitis, Crohn's disease, multiple sclerosis (MS), systemic lupuserythematosus (SLE), graft versus host disease (GVHD), rheumatoidarthritis, or psoriasis.

In some embodiments, the IL-10 comprises at least 80%, 85%, 90%, 92%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acidsequence set forth in SEQ ID NO: 2. In some embodiments, the one or morepharmaceutically acceptable excipients comprise a surfactant. In someembodiments, the surfactant is selected from the group consisting of:polysorbate 80, polysorbate 20, and poloxamer 188. In some embodiments,the one or more pharmaceutically acceptable excipients comprise anosmolyte. In some embodiments, the osmolyte is selected from the groupconsisting of sucrose and trehalose. In some embodiments, the one ormore pharmaceutically acceptable excipients comprise a salt. In someembodiments, the salt is selected from the group consisting of potassiumphosphate, sodium chloride, potassium chloride, magnesium chloride, andsodium sulfate. In some embodiments, the one or more pharmaceuticallyacceptable excipients comprise a bulking agent. In some embodiments, thebulking agent is selected from the group consisting of: glycine andmannitol. In some embodiments, the one or more pharmaceuticallyacceptable excipients comprise a disintegrant. In some embodiments, thedisintegrant is selected from the group consisting of: microcrystallinecellulose (MCC), silicified microcrystalline cellulose (SMCC), starch,sodium starch glycolate, veegum, bentonite, alginic acid, calciumalginate, croscarmellose sodium (crosslinked sodium carboxymethylcellulose), and crospovidone (crosslinked polyvinylpyrrolidone). In someembodiments, the one or more pharmaceutically acceptable excipientscomprise a binding agent. In some embodiments, the binding agent isselected from the group consisting of: sucrose, lactose, starch,cellulose, gelatin, polyvinylpyrrolidone (PVP), and polyethylene glycol(PEG). In some embodiments, the one or more pharmaceutically acceptableexcipients comprise a lubricant. In some embodiments, the lubricant isselected from the group consisting of: magnesium stearate, glycerylbehenate, glyceryl dibehenate, sodium stearyl fumerate, stearic acid,talc, silica, calcium stearate, magnesium carbonate, hydrogenated oil,mineral oil, polyethylene glycol (PEG), and glyceryl monostearate.

In some embodiments, the IL-10 is part of an IL-10 delivery constructcomprising the IL-10 coupled to a carrier. In some embodiments, theIL-10 delivery construct comprises a linker coupling the IL-10 to thecarrier. In some embodiments, the linker comprises an amino acidsequence set forth in SEQ ID NO: 6. In some embodiments, the carrier hasat least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to an amino acid sequence set forth in SEQ ID NO: 4.In some embodiments, the IL-10 delivery construct has at least 80%, 85%,90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to anamino acid sequence set forth in SEQ ID NO: 5. In some embodiments, uponcontact with a cell, the carrier promotes endocytosis or transcytosis ofthe IL-10 delivery construct. In some embodiments, the cell is a gutepithelial cell. In some embodiments, the gut epithelial cell is apolarized gut epithelial cell.

In some embodiments, the formulation is an oral formulation. In someembodiments, the oral formulation is a capsule or a tablet. In someembodiments, the oral formulation comprises a first coating comprising afirst copolymer and a second copolymer, wherein the first coating isexternal of the IL-10 and one or more pharmaceutically acceptableexcipients. In some embodiments, the first copolymer comprisesmethacrylic acid and ethyl acrylate. In some embodiments, the secondcopolymer comprises methacrylic acid, methyl methacrylate, and methylacrylate. In some embodiments, a ratio of the first copolymer to thesecond copolymer in the first coat is from 0.8:1 to 1.2:1. In someembodiments, the administering comprises oral administration.

In some embodiments the present disclosure provides a method of treatingan inflammatory disease in a subject in need thereof, the methodcomprising orally administering an IL-10 therapeutic to the subject andadministering a non-IL-10 immunosuppressor to the subject. In otherembodiments the present disclosure provides a method of treating aninflammatory disease in a subject in need thereof, the method comprisingorally administering an IL-10 therapeutic to the subject, wherein thesubject concomitantly receives a non-IL-10 immunosuppressor. In furtherembodiments the present disclosure provides a method of treating aninflammatory disease in a subject, wherein the subject had an inadequateresponse to anon-IL-10 immunosuppressor, the method comprising orallyadministering an IL-10 therapeutic to the subject.

In some cases, the method further comprises administering the non-IL-10immunosuppressor with the IL-10 therapeutic. In some cases, the subjectwas treated with the non-IL-10 immunosuppressor for at least 6 weeksprior to determining the inadequate response. In some cases, the subjectwas treated with the non-IL-10 immunosuppressor for at least 12 weeksprior to determining the inadequate response. In some cases, theinadequate response is a partial response.

In some cases, the inflammatory disease is selected from the groupconsisting of: inflammatory bowel disease, psoriasis, plaque psoriasis,hidradenitis suppurativa, psoriatic arthritis, rheumatoid arthritis,juvenile idiopathic arthritis, ankylosing spondylitis, bacterial sepsis,Crohn's disease, fistulizing Crohn's disease, moderate-to-severeulcerative colitis, mild-to-moderate ulcerative colitis, ulcerativecolitis, collagenous colitis, lymphocytic colitis, ischaemic colitis,diversion colitis, Behcet's syndrome, indeterminate colitis,pancreatitis, liver inflammation, pouchitis, proctitis, uveitis, graftvs host disease, and epithelial cell injury. In some cases, theinflammatory disease is an inflammatory bowel disease. In some cases,the inflammatory disease is selected from the group consisting of:rheumatoid arthritis, ulcerative colitis, and Crohn's disease.

In some cases, the inflammatory disease is rheumatoid arthritis and thesubject with an inadequate response has one or more joints with activedisease. In some cases, the one or more joints with active disease areidentified by fluorescent optical imaging or magnetic resonance imaging.In some cases, the subject with an inadequate response additionally hastwo or more joints which are tender. In some cases, the subject with aninadequate response additionally has two or more joints which areswollen.

In some cases, the inflammatory disease is ulcerative colitis, and thesubject with an inadequate response has moderate to severe ulcerativecolitis. In some cases, the subject with an inadequate response has amodified Mayo Clinic Score (MMS) of between about 4 points and about 9points. In some cases, the subject with an inadequate response has acentrally read MCS endoscopic sub score of grade 2 or higher. In somecases, the subject with an inadequate response has a MMS rectal bleedingsub score of 1 point or higher. In some cases, the subject with aninadequate response has disease extending 15 cm or more from the analverge. The method of any one of the above claims, wherein the IL-10therapeutic is an IL-10 delivery construct.

In some cases, the IL-10 delivery construct comprises a carrierconsisting of an amino acid sequence set forth in SEQ ID NO: 4. In somecases, the IL-10 delivery construct comprises a carrier consisting of anamino acid sequence at least 90% identical to SEQ ID NO: 4. In somecases, the IL-10 delivery construct comprises an amino acid sequence setforth in SEQ ID NO: 5.

In some cases, the non-IL-10 immunosuppressor is a TNF alpha inhibitor.In some cases, the TNF alpha inhibitor is a monoclonal antibody. In somecases, the TNF alpha inhibitor is selected from the group consisting ofinfliximab (Remicade), adalimumab (Humira) and golimumab (Simponi). Insome cases, the TNF alpha inhibitor comprises SEQ ID NO: 151 and SEQ IDNO: 152. In some cases, the TNF alpha inhibitor comprises SEQ ID NO: 153and SEQ ID NO: 154. In some cases, the TNF alpha inhibitor is notetanercept.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. Various features of the disclosure are set forthwith particularity in the appended claims. A better understanding of thefeatures and advantages of the present disclosure will be obtained byreference to the following detailed description that sets forthillustrative embodiments, in which the principles of the disclosure areutilized, and the accompanying drawings of which:

FIG. 1 illustrates the structure of a cholix-IL-10 delivery constructhomodimer (a dimer comprising two identical subunits of SEQ ID NO: 5) asdetermined by small angle X-ray scattering (SAXS).

FIGS. 2A-2B illustrate an exemplary process for expressing, refolding,and purifying IL-10 or IL-10 delivery constructs. FIG. 2A illustrates anexemplary process for expressing, refolding, and purifying IL-10 orIL-10 delivery constructs with a sulfitolysis step. FIG. 2B illustratesan exemplary process for expressing, refolding, and purifying IL-10 orIL-10 delivery constructs without a sulfitolysis step.

FIG. 3 illustrates an adapted SHIME® system simulating the physiologicalconditions of stomach, small intestine, and colon within the samereactor over time.

FIG. 4 illustrates a pH profile of a simulated GI tract under fastedconditions. Arrows indicate the time and corresponding pH of samplestaken during the stomach incubation phase (ST0; ST45) and smallintestine incubation phase (SI0; SI0,5; SI1; SI1,5; SI2; SI3).

FIGS. 5A-5E illustrate average release of caffeine (mg) from size 1capsules with various coating compositions and coating thickness, asshown by the capsule formulations in TABLE 12. Average release wasdetermined from 3 individual capsules. Time points with conditionssimulating the stomach are represented by ST0 and ST45. Time points withconditions simulating the small intestine are represented by SI0, SI0,5,SI1, SI1,5, SI2, and SI3. Time points with conditions simulating thecolon are represented by C0, C0,5, C1, C1,5, C2, C3, C4, and C18. FIG.5A illustrates release of caffeine from a capsule of formulation A.Differences in samples as compared to their preceding sample areindicated with an asterisk (*), which represents a statisticallysignificant change (p<0.05). The visual scores of the capsules areindicated above the bars (1: capsule intact; 2: capsule damaged butalmost all product is still in the capsule; 3: capsule damaged and allproduct is released; 4: capsule destroyed). FIG. 5B illustrates releaseof caffeine from a capsule of formulation B. FIG. 5C release of caffeinefrom a capsule of formulation C. FIG. 5D illustrates release of caffeinefrom a capsule of formulation D. FIG. 5E illustrates release of caffeinefrom a capsule of formulation E.

FIGS. 6A-6C illustrate percent caffeine release from various capsulecoatings, the first hour at pH 1.0 and the remaining time at pH 7.0.FIG. 6A illustrates percent caffeine release from capsule coatings A-B.FIG. 6B illustrates percent caffeine release from capsule coatings C-F.FIG. 6C illustrates percent caffeine release from capsule coatings G-H.Capsule coatings A-H are described in TABLE 23.

FIGS. 7A-7C illustrate percent caffeine release from various capsulecoatings, the first hour at pH 1.0 and the remaining time at pH 6.5.FIG. 7A illustrates percent caffeine release from capsule coatings A-B.FIG. 7B illustrates percent caffeine release from capsule coatings C-F.FIG. 7C illustrates percent caffeine release from capsule coatings G-H.Capsule coatings A-H are described in TABLE 23.

FIGS. 8A-8C illustrate percent caffeine release from various capsulecoatings, the first hour at pH 1.0 and the remaining time at pH 6.0.FIG. 8A illustrates percent caffeine release from capsule coatings A-B.FIG. 8B illustrates percent caffeine release from capsule coatings C-F.FIG. 8C illustrates percent caffeine release from capsule coatings G-H.Capsule coatings A-H are described in TABLE 23.

FIGS. 9A-9C illustrate percent target construct (SEQ ID NO: 5) releasefrom various capsule coatings, the first hour at pH 1.0 and theremaining time at pH 7.0. FIG. 9A illustrates percent target constructrelease from capsule coatings A-B. FIG. 9B illustrates percent targetconstruct release from capsule coatings C-F. FIG. 9C illustrates percenttarget construct release from capsule coatings G-H. Capsule coatings A-Hare described in TABLE 23.

FIGS. 10A-10C illustrate percent target construct (SEQ ID NO: 5) releasefrom various capsule coatings, the first hour at pH 1.0 and theremaining time at pH 6.5. FIG. 10A illustrates percent target constructrelease from capsule coatings A-B. FIG. 10B illustrates percent targetconstruct release from capsule coatings C-F. FIG. 10C illustratespercent target construct release from capsule coatings G-H. Capsulecoatings A-H are described in TABLE 23.

FIGS. 11A-11C illustrate percent target construct (SEQ ID NO: 5) releasefrom various capsule coatings, the first hour at pH 1.0 and theremaining time at pH 6.0. FIG. 11A illustrates percent target constructrelease from capsule coatings A-B. FIG. 11B illustrates percent targetconstruct release from capsule coatings C-F. FIG. 11C illustratespercent target construct release from capsule coatings G-H. Capsulecoatings A-H are described in TABLE 23.

FIGS. 12A-12C illustrate percent released target constructs (SEQ ID NO:5) in the dimer form from various capsule coatings, the first hour at pH1.0 and the remaining time at pH 7.0. FIG. 12A illustrates percentreleased target constructs in the dimer form from capsule coatings A-B.FIG. 12B illustrates percent released target constructs in the dimerform from capsule coatings C-F. FIG. 12C illustrates percent releasedtarget constructs in the dimer form from capsule coatings G-H. Capsulecoatings A-H are described in TABLE 23.

FIGS. 13A-13C illustrate percent released target constructs (SEQ ID NO:5) in the dimer form from various capsule coatings, the first hour at pH1.0 and the remaining time at pH 6.5. FIG. 13A illustrates percentreleased target constructs in the dimer form from capsule coatings A-B.FIG. 13B illustrates percent released target constructs in the dimerform from capsule coatings C-F. FIG. 13C illustrates percent releasedtarget constructs in the dimer form from capsule coatings G-H. Capsulecoatings A-H are described in TABLE 23.

FIGS. 14A-14C illustrate percent released target constructs (SEQ ID NO:5) in the dimer form from various capsule coatings, the first hour at pH1.0 and the remaining time at pH 6.0. FIG. 14A illustrates percentreleased target constructs in the dimer form from capsule coatings A-B.FIG. 14B illustrates percent released target constructs in the dimerform from capsule coatings C-F. FIG. 14C illustrates percent releasedtarget constructs in the dimer form from capsule coatings G-H. Capsulecoatings A-H are described in TABLE 23.

FIGS. 15A-15C illustrate serum levels in cynomolgus monkeys of IL-10,caffeine, and interleukin-1 receptor antagonist (IL-IRA) during the 8hours following administration to the monkeys of capsules containing atarget construct (SEQ ID NO: 5) and caffeine with one of capsulecoatings A, B, or C as shown in TABLE 25. FIG. 15A illustrates serumlevels of IL-10. FIG. 15B illustrates serum levels of caffeine. FIG. 15Cillustrates serum levels of IL-IRA. X and Y axes are a log scale. Meanfor each group is plotted with bars representing standard error of themean.

FIGS. 16A-16C illustrate serum levels in cynomolgus monkeys of IL-10,caffeine, and IL-IRA during the 8 hours following administration to themonkeys of capsules containing a target construct (SEQ ID NO: 5) andcaffeine with one of capsule coatings A, G, and H as shown in TABLE 25.FIG. 16A illustrates serum levels of IL-10. FIG. 16B illustrates serumlevels of caffeine. FIG. 16C illustrates serum levels of IL-IRA. X and Yaxes are a log scale. Mean for each group is plotted with barsrepresenting standard error of the mean.

FIGS. 17A-17C illustrate serum levels in cynomolgus monkeys of IL-10,caffeine, and IL-IRA during the 8 hours following administration to themonkeys of capsules containing a target construct (SEQ ID NO: 5) andcaffeine with one of capsule coatings A, C, D, E, and F as shown inTABLE 25. FIG. 17A illustrates serum levels of IL-10. FIG. 17Billustrates serum levels of caffeine. FIG. 17C illustrates serum levelsof IL-IRA. X and Y axes are a log scale. Mean for each group is plottedwith bars representing standard error of the mean.

FIG. 18 illustrates a size exclusion chromatogram (SEC) of combinationsof different compacting excipients and a lyophilized target construct(SEQ ID NO: 5) powder after being incubated at 40° C. for 3 days.Compacting excipients examined included: starch, croscarmellose sodium,magnesium stearate, glyceryl behenate, microcrystalline cellulose (MCC),lactose, crospovidone, and silicified microcrystalline cellulose (SMCC).

FIG. 19 illustrates a size exclusion chromatogram (SEC) showing targetconstruct (SEQ ID NO: 5) dimer purity as well as dimer purity of thetarget construct (SEQ ID NO: 5) of the F1 and F2 formulations.

FIGS. 20A-20D illustrate total recovery of the target construct (SEQ IDNO: 5), recovery of the dimer, and dimer percentage followingdissolution of different tablet formulations. FIG. 20A illustrates totalrecovery of the target construct, recovery of the dimer, and dimerpercentage following dissolution of an F1 tablet created using acompression force of 2000 pound-force (lbf). FIG. 20B illustrates totalrecovery of the target construct, recovery of the dimer, and dimerpercentage following dissolution of an F1 tablet created using acompression force 2500 lbf. FIG. 20C illustrates total recovery of thetarget construct, recovery of the dimer, and dimer percentage followingdissolution of an F2 tablet created using a compression force of 2500lbf. FIG. 20D illustrates total recovery of the target construct,recovery of the dimer, and dimer percentage following dissolution of anF2 tablet created using a compression force of 3000 lbf. In theseexperiments, dimer recovery indicated the absolute amount of dimeridentified relative to a reference standard. In these experiments, dimerpurity indicated the percent of dimer relative to all forms of the IL-10delivery construct detected (which included aggregates and monomers).Analysis was carried out at pH 7.0.

FIG. 21 illustrates percent of target constructs (SEQ ID NO: 5) in thedimer form in different lyophilization formulations before and after a25° C. incubation. The horizontal line indicates the main peak dimerpurity for the reference sample (1×PBS—no excipients) after 3 days at25° C.

FIG. 22 illustrates percent of target constructs (SEQ ID NO: 5) in thedimer form in different lyophilization formulations before and after 5freeze/thaw cycles (F/T) at −20° C.

FIGS. 23A-23B illustrate the effect of 5 freeze/thaw cycles, at −20° C.and −80° C., on target constructs (SEQ ID NO: 5) aggregates and dimers.FIG. 23A illustrate the effect of 5 freeze/thaw cycles on the targetconstruct aggregate (HMW) percentage. FIG. 23B illustrate the effect of5 freeze/thaw cycles on the target constructs dimer percentage.

FIGS. 24A-24B illustrate the change in percent of target aggregates ordimers at 4° C. or 25° C. over a time course of one week in differentformulations of lyophilization buffer from TABLE 11. Two differentconcentrations of target constructs (SEQ ID NO: 5) (20 mg/ml and 40mg/ml) in the lyophilization buffers were examined for each of the fourdifferent formulations. FIG. 24A illustrates the change in percent oftarget construct aggregates at 4° C. FIG. 24B illustrates the change inpercent of target dimer at 4° C. FIG. 24C illustrates the change inpercent of target construct aggregates at 25° C. Arrows indicate thelyophilization buffer containing sucrose at pH 7.5. FIG. 24D illustratesthe change in percent of target construct dimer at 25° C. Arrowsindicate the lyophilization buffer containing sucrose at pH 7.5.

FIGS. 25A-25B illustrate refolding efficiency when varying arginineconcentration and target construct (SEQ ID NO: 5) concentration of therefolding solution. FIG. 25A shows a contour plot of refoldingefficiency (% of dimer at end of refolding). FIG. 25B shows a bar plotof refolding efficiencies.

FIGS. 26A-26B illustrate refolding efficiency of the target construct(SEQ ID NO: 5) when varying glycerol concentration and pH of therefolding solution. FIG. 26A shows a contour plot of refoldingefficiency. FIG. 26B shows a bar plot of refolding efficiencies.

FIGS. 27A-27B illustrate refolding efficiency of the target construct(SEQ ID NO: 5) when varying sucrose concentration and PEG 3350concentration of the refolding solution. FIG. 27A shows a contour plotof refolding efficiency. FIG. 27B shows a bar plot of refoldingefficiencies.

FIG. 28 illustrates a size exclusion high performance liquidchromatography (SE-HPLC) chromatogram showing target construct (SEQ IDNO: 5) aggregates, dimers, and monomers for each of four refoldingsolutions. “A” represents the control refolding solution containing 0.7M arginine. “B” represents a refolding solution with 1M arginine. “C”represents a refolding solution with 1M arginine plus 0.25 M sucroseplus 0.2% PEG3350. “D” represents 1M arginine plus 0.25M sucrose.

FIG. 29 illustrates refolding efficiency of each of the four refoldingsolutions illustrated in FIG. 28.

FIG. 30 illustrates Coomassie blue staining of target constructs atvarious intermediate steps in the purification process followingSDS-PAGE. Lanes 1 and 11 contain mark 12 molecular weight markers. Lanes8, 9, 10, 18, 19, and 20 are blank. The samples in lanes 2 through 10SDS-PAGE were run in reduced conditions. The samples in lanes 12 through20 were SDS-PAGE run in non-reduced conditions. Lanes 2 and 12 containthe target construct (SEQ ID NO: 5). Lanes 3 and 13 contain filteredTFF-2 retentate (Cycle #1). Lanes 4 and 14 contains filtered TFF-2retentate (Cycle #2). Lanes 5 and 15 contains Capto™ Q pooled eluate.Lanes 6 and 16 contains the CHT pooled eluate. Lanes 7 and 17 containsthe TFF-3 final retentate.

FIGS. 31A-31B illustrate embodiments of oral formulations 3200 and 3205described herein. FIG. 31A illustrates an oral formulation 3200comprising an interior region comprising therapeutic protein (3201), afirst coat (3203), a second coat (3202), and a third coat (3204). FIG.31B illustrates an oral formulation comprising a first coat (3203).

FIGS. 32A-32B illustrate time of radiolabel release from capsules with acoating of formulation 1, 2, or 3, as described in TABLE 35. FIG. 32Aillustrates time of initial radiolabel release following administrationof oral capsule coating formulations in healthy volunteers. FIG. 32Billustrates time of complete radiolabel release following administrationof oral capsule coating formulations in healthy volunteers.

FIGS. 33A-33B illustrate anatomical location of radiolabel release fromcapsules with a coating of formulation 1, 2, or 3, as described in TABLE35. FIG. 33A illustrates anatomical location of initial radiolabelrelease following administration of oral capsule coating formulations inhealthy volunteers. FIG. 33B illustrates anatomical location of completeradiolabel release following administration of oral capsule coatingformulations in healthy volunteers. PSB=proximal small bowel; DSB=distalsmall bowel; AC=ascending colon; TC=transverse colon; DC=descendingcolon

FIG. 34 illustrates a size exclusion chromatogram (SEC) identifyingpeaks representing target construct (SEQ ID NO: 5) dimer, aggregate, andmonomer.

FIG. 35 illustrates the effect of the IL-10 delivery construct (SEQ IDNO: 5) on percentage change in body weight in mice followingoxazolone-induced colonic inflammation. Body weight was recorded dailyin mice preceding and following the insult. Data are expressed asmean±SEM; n per group: naive (5), vehicle (10), IL-10 delivery construct(15), 5-ASA (15). Data were analyzed by 2-way ANOVA with Dunnett'spost-hoc test to compare difference of each group vs. vehicle at eachday. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

FIG. 36 illustrates the effect of the IL-10 delivery construct (SEQ IDNO: 5) on survival rates in mice following oxazolone-induced colonicinflammation. Mortality was recorded daily in mice preceding andfollowing the insult. Data are expressed as percentage survival.

FIG. 37 illustrates the effect of the IL-10 delivery construct (SEQ IDNO: 5) on disease severity in mice following oxazolone-induced colonicinflammation. Severity was assessed by colonic markers of inflammation 7days after the insult.

FIG. 38A illustrates the effect of the IL-10 delivery construct (SEQ IDNO: 5) on colon weight in mice following oxazolone-induced colonicinflammation. Colon weight was measured 7 days after the insult.

FIG. 38B illustrates the effect of the IL-10 delivery construct (SEQ IDNO: 5) on hemoccult positivity in mice following oxazolone-inducedcolonic inflammation.

FIG. 38C illustrates the effect of the IL-10 delivery construct (SEQ IDNO: 5) on stool consistency in mice following oxazolone-induced colonicinflammation.

FIG. 38D illustrates the effect of the IL-10 delivery construct (SEQ IDNO: 5) on disease activity index in mice following oxazolone-inducedcolonic inflammation.

FIG. 38E illustrates the effect of the IL-10 delivery construct (SEQ IDNO: 5) on serum levels of macrophage colony-stimulating factor 1 (MCSF)in mice following oxazolone-induced colonic inflammation.

FIG. 38F illustrates the effect of the IL-10 delivery construct (SEQ IDNO: 5) on serum levels of IL12 p70 protein in mice followingoxazolone-induced colonic inflammation.

FIG. 38G illustrates the effect of the IL-10 delivery construct (SEQ IDNO: 5) on serum levels of IL-3 in mice following oxazolone-inducedcolonic inflammation.

FIGS. 39A-39E illustrates effects of oral IL-10 delivery constructadministration on cellular expression of proteins relevant to theinflammatory processes associated with ulcerative colitis.Cross-sections from the proximal, mid, and distal colon from micefollowing oxazolone-induced colonic inflammation were analyzed byimmunohistochemistry. FIG. 39A illustrates the effect of oral IL-10delivery construct administration on cellular expression of NFκB. FIG.39B illustrates the effect of oral IL-10 delivery constructadministration on cellular expression of TNFα. FIG. 39C illustrates theeffect of oral IL-10 delivery construct administration on cellularexpression of CD4. FIG. 39D illustrates the effect of oral IL-10delivery construct administration on cellular expression of IL-4. FIG.39E illustrates the effect of oral IL-10 delivery constructadministration on cellular expression of Foxp3.

FIGS. 40A-40B illustrate a Luminex array of systemic cytokines followingoral delivery of an IL-10 delivery construct dosing solution. FIG. 40Aillustrates a Luminex array of IL-6 following oral delivery of an IL-10delivery construct dosing solution. FIG. 40B illustrates a Luminex arrayof IL-23 following oral delivery of an IL-10 delivery construct dosingsolution.

FIGS. 41A-41J illustrate concentration of 10 cytokines in plasma samplesusing MSD Proinflammatory Panel 1 following the indicated treatments.FIG. 41A illustrates plasma concentration of IFNγ. FIG. 41B illustratesplasma concentration of IL-10. FIG. 41C illustrates plasma concentrationof IL-12p70. FIG. 41D illustrates plasma concentration of IL-10. FIG.41E illustrates plasma concentration of IL-2. FIG. 41F illustratesplasma concentration of IL-4. FIG. 41G illustrates plasma concentrationof IL-5. FIG. 41H illustrates plasma concentration of IL-6. FIG. 41Iillustrates plasma concentration of KC/GRO. FIG. 41J illustrates plasmaconcentration of TNF-α.

FIG. 42 illustrates the effect of the IL-10 delivery construct of SEQ IDNO. 5 on percentage change in body weight in mice followingoxazolone-induced inflammatory colitis.

FIG. 43 illustrates the effect of the IL-10 delivery construct of SEQ IDNO. 5 on percentage survival in mice following oxazolone-inducedinflammatory colitis. Mortality was recorded daily in mice preceding andfollowing the oxazolone insult. Data are expressed as percentagesurvival.

FIG. 44 illustrates the effect of the IL-10 delivery construct of SEQ IDNO. 5 on disease severity in mice following oxazolone-induced colonicinflammation. Disease activity index (DAI) was scored by fecalconsistency and hemoccult positivity following the oxazolone insult.Data are expressed as mean±SEM.

FIG. 45 illustrates the effect of the IL-10 delivery construct of SEQ IDNO. 5 on colon weight/length ratio in mice following oxazolone-inducedcolonic inflammation. Colon weight and length were measured 7 days afterthe oxazolone insult. Data are expressed as mean±SEM.

FIG. 46 illustrates the histopathology of the proximal, mid, and distalcolon following oxazolone-induced colonic inflammation in mice. Data areexpressed as mean±SEM.

FIGS. 47A-47LL illustrate systemic concentrations of circulatingcytokines, chemokines, and growth factors in mice. Plasma concentrationsof circulating cytokines were analyzed using the Luminex bead array.Data are expressed as mean±SEM. FIG. 47A illustrates systemicconcentration of GCSF/CSF3. FIG. 47B illustrates systemic concentrationof GMCSF. FIG. 47C illustrates systemic concentration of MCSF. FIG. 47Dillustrates systemic concentration of VEGF. FIG. 47E illustratessystemic concentration of LIF. FIG. 47F illustrates systemicconcentration of Exotaxin. FIG. 47G illustrates systemic concentrationof GROA. FIG. 47H illustrates systemic concentration of IP10. FIG. 47Iillustrates systemic concentration of LIX. FIG. 47J illustrates systemicconcentration of MCP1. FIG. 47K illustrates systemic concentration ofMCP3. FIG. 47L illustrates systemic concentration of MIP1α. FIG. 47Millustrates systemic concentration of MIP1β. FIG. 47N illustratessystemic concentration of MIP2. FIG. 47O illustrates systemicconcentration of RANTES. FIG. 47P illustrates systemic concentration ofIL-la. FIG. 47Q illustrates systemic concentration of IL-10. FIG. 47Rillustrates systemic concentration of IL-2. FIG. 47S illustratessystemic concentration of IL-3. FIG. 47T illustrates systemicconcentration of IL-4. FIG. 47U illustrates systemic concentration ofIL-5. FIG. 47V illustrates systemic concentration of IL-6. FIG. 47Willustrates systemic concentration of IL-9. FIG. 47X illustratessystemic concentration of IL-12p70. FIG. 47Y illustrates systemicconcentration of IL-13. FIG. 47Z illustrates systemic concentration ofIL-15/IL-15R. FIG. 47AA illustrates systemic concentration of IL-17A.FIG. 47BB illustrates systemic concentration of IL-18. FIG. 47CCillustrates systemic concentration of IL-23. FIG. 47DD illustratessystemic concentration of IL-27. FIG. 47EE illustrates systemicconcentration of IL-28. FIG. 47FF illustrates systemic concentration ofIL-31. FIG. 47GG illustrates systemic concentration of IFN-α. FIG. 47HHillustrates systemic concentration of IFN-γ. FIG. 47II illustratessystemic concentration of TNF-α. FIG. 47JJ illustrates systemicconcentration of IL-10. FIG. 47KK illustrates systemic concentration ofIL-22. FIG. 47LL illustrates systemic concentration of TGF-β.

FIGS. 48A-48J illustrate concentrations of 10 cytokines in plasmasamples using V-PLEX proinflammatory panel. FIG. 48A illustrates plasmaconcentration of IFNγ. FIG. 48B illustrates plasma concentration ofIL-10. FIG. 48C illustrates plasma concentration of IL-12p70. FIG. 48Dillustrates plasma concentration of IL-10. FIG. 48E illustrates plasmaconcentration of IL-2. FIG. 48F illustrates plasma concentration ofIL-4. FIG. 48G illustrates plasma concentration of IL-5. FIG. 48Hillustrates plasma concentration of IL-6. FIG. 48I illustrates plasmaconcentration of KC/GRO. FIG. 48J illustrates plasma concentration ofTNF-α. Data are expressed as mean±SEM.

FIGS. 49A-49D illustrate systemic and colonic IL-1Ra expression in micefollowing oxazolone-induced inflammatory colitis. FIG. 49A illustratessystemic plasma concentration of IL-1Ra. FIG. 49B illustrates geneexpression of IL-1Ra in colonic tissue of naive, vehicle, and 9 mg/kgIL-10 delivery construct treated mice. FIG. 49C illustrates geneexpression of IL-10 in colonic tissue of naive, vehicle, and 9 mg/kgIL-10 delivery construct of SEQ ID NO. 5 treated mice. FIG. 49Dillustrates the IL-1Ra/IL-10 ratio. mRNA transcript levels werenormalized to GAPDH. Data are expressed as mean±SEM.

FIG. 50A illustrates the timeline of dextran sulfate sodium(DSS)-induction of colitis and treatment with daily oral gavage of theIL-10 delivery construct of SEQ ID NO. 5 (as designated) dissolved in100 mL of PBS on days designated by a downward arrow.

FIG. 50B illustrates DSS-induced weight loss during the in-life portionof the study.

FIG. 50C illustrates the effect of the IL-10 delivery construct of SEQID NO. 5 on body weight following DSS-induced colitis. Body weightpresented as percentage change from baseline following DSS-inducedinflammation. Data are expressed as mean±SEM.

FIG. 51 illustrates the effect of the IL-10 delivery construct of SEQ IDNO. 5 on disease activity index (DAI) following DSS-induced colitis.Individual scores for weight loss, stool consistency, and stoolhemoccult (scored 0-3) were summed to provide a DAI (0-9 range) inresponse to DSS-induced inflammation. Data are expressed as mean±SEM.

FIGS. 52A-B illustrate the effect of the IL-10 delivery construct of SEQID NO. 5 on colon length (FIG. 52A) and weight (FIG. 52B) followingDSS-induced colitis. Data are expressed as mean±SEM.

FIG. 53 illustrates the effect of the IL-10 delivery construct of SEQ IDNO. 5 on summed histology parameters (inflammation, gland loss, erosion,and hyperplasia) following DSS-induced colitis. Data are expressed asmean±SEM. ****p<0.0001, *p<0.05.

FIG. 54 illustrates the effect of the IL-10 delivery construct of SEQ IDNO. 5 on edema width following DSS-induced colitis. Data are expressedas mean±SEM. ****p<0.0001, **p<0.01, *p<0.05.

FIG. 55 illustrates the effect of the IL-10 delivery construct of SEQ IDNO. 5 on colonic mucosal thickness following DSS-induced colitis. Dataare expressed as mean±SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

FIG. 56 illustrates the effect of the IL-10 delivery construct of SEQ IDNO. 5 on colonic hyperplasia following DSS-induced colitis. Data areexpressed as mean±SEM. ****p<0.0001, **p<0.01.

FIGS. 57A-57B illustrate variable human IL-10 detection and IL-1Rainduction in the DSS study. Systemic concentrations were measured bysandwich immunoassays following DSS insult. Data are expressed asmean±SEM. FIG. 57A illustrates systemic concentrations of the IL-10delivery construct, as detected by anti-cholix or anti-IL-10 detectionantibodies. FIG. 57B illustrates systemic concentrations of IL-1Rafollowing DSS insult.

FIG. 58 illustrates plasma concentration of total IL-10 in non-humanprimates (NHPs) post-dose with IL-10 delivery construct (SEQ ID NO: 5).Systemic concentrations of total IL-10 measured by immunoassay followingoral administration of IL-10 delivery construct capsules. Data areexpressed as mean±SEM.

FIG. 59 illustrates plasma concentration of IL-1Ra in NHPs post-dosewith the IL-10 delivery construct (SEQ ID NO: 5). Systemicconcentrations of IL-1Ra measured by immunoassay following oraladministration of IL-10 delivery construct capsules. Data are expressedas mean±SEM.

FIG. 60 illustrates plasma concentration of caffeine in NHPs pose-dosewith the IL-10 delivery construct (SEQ ID NO: 5). Systemicconcentrations of caffeine measured by immunoassay following oraladministration of IL-10 delivery construct capsules. Data are expressedas mean±SEM.

FIGS. 61A-61E illustrate plasma concentrations of selectedproinflammatory cytokines in NHPs after oral dosing with the IL-10delivery construct (SEQ ID NO: 5). FIG. 61A illustrates plasmaconcentration of IFNγ. FIG. 61B illustrates plasma concentration ofIL-10. FIG. 61C illustrates plasma concentration of IL-2. FIG. 61Dillustrates plasma concentration of IL-8. FIG. 61E illustrates plasmaconcentration of IL-6.

FIG. 62 illustrates that the IL-10 delivery construct showed little orno co-localization with LAMP1-positive lysosomes in enterocytes over a15-minute time course study.

FIG. 63 illustrates an immuno-fluorescence image of CD11c containingcells (dendritic cells) and IL-10.

FIG. 64 illustrates an immuno-fluorescence image of CD19 containingcells (B lymphocytes) and IL-10.

FIG. 65 illustrates an immuno-fluorescence image of CD34 containingcells (endothelia) and cholix.

FIG. 66 illustrates an immuno-fluorescence image of CD3 containing cells(T lymphocytes) and IL-10.

FIG. 67 illustrates cellular targeting of the IL-10 delivery constructto T cells and macrophages in the GI submucosa.

FIG. 68 illustrates dissolution of coated capsules containing the IL-10delivery construct in a Type 4 dissolution apparatus. Symbol key:squares: HPMC sub coating only; circles: HPMC sub coating plus eudragit50:50 coating for 80 min; plus signs: HPMC sub coating plus eudragit50:50 coating for 120 min; diamonds: HPMC sub coating plus eudragit50:50 coating for 120 min plus HPMC coating for 20 min; and triangles:HPMC sub coating plus eudragit 50:50 coating for 120 min plus HPMCcoating for 60 min.

FIG. 69 illustrates dissolution of coated tablets containing the IL-10delivery construct in a Type 4 dissolution apparatus.

FIG. 70 illustrates recovery of dimer forms of the IL-10 deliveryconstruct (lower section of each bar) as well as monomer (LMW) andaggregate (HMW) forms (upper section of each bar) of the IL-10 deliveryconstruct across the full time course shown in FIG. 68 and FIG. 69. Dataillustrates the area-under-the-curve from t=0 to the last time pointmeasured. From left to right, bars represent: (1) non-coated tablet (2)tablet with 8 mg coat weight of 20:80 weight ratio of Eudragit® L30D55:Eudragit® FS30D; (3) tablet with 13 mg coat weight of 20:80 weight ratioof Eudragit® L30D55: Eudragit® FS30D; (4) tablet with 20 mg coat weightof 20:80 weight ratio of Eudragit® L30D55: Eudragit® FS30D; (5) tabletwith 8 mg coat weight of 50:50 weight ratio of Eudragit® L30D55:Eudragit® FS30D; (6) tablet with 13 mg coat weight of 50:50 weight ratioof Eudragit® L30D55: Eudragit® FS30D; (7) tablet with 8 mg coat weightof 50:50 weight ratio of Eudragit® L30D55: Eudragit® FS30D; (8)Enteric-No; (9) Enteric-80m; (10) Enteric-120m; (11) Enteric120m+HPMC60m; (12) Enteric 120m+HPMC20m.

FIGS. 71A-71C illustrate systemic concentrations of certain markersmeasured over 24 hours by immunoassay, following pan-colonicadministration of IL-10 delivery construct (SEQ ID NO: 5) at 1, 3, and10 mg (n=3/group). Data are expressed as mean±SEM, statistical analysisnot performed. FIG. 71A illustrates systemic concentration of IL-10.FIG. 71B illustrates systemic concentration of IL-10 delivery construct(SEQ ID NO: 5). FIG. 71C illustrates systemic concentration of IL-1Ra.

FIG. 72 illustrates systemic concentration of IL-6 measured over 24hours by immunoassay, following pan-colonic administration of IL-10delivery construct (SEQ ID NO: 5) at 1, 3, and 10 mg (n=3/group). Dataare expressed as mean±SEM, statistical analysis not performed.

FIG. 73 illustrates concentration of IL-1Ra measured over 24 h byimmunoassay, following pan-colonic administration of IL-10 deliveryconstruct (SEQ ID NO: 5) at 1, 3 and 10 mg. Data are expressed asmean±SEM; n per IL-10 delivery construct dose: predose (2), 15 min (3),30 min (3), 45 min (3), 8 h (1) and 24 h (1); statistical analysis notperformed.

FIG. 74 illustrates STAT3 phosphorylation in colonic tissue, as measuredby the ratio of pSTAT3 to total STAT3. Phosphorylation and totalexpression were measured by immunoassay over 45 min, followingpan-colonic administration of IL-10 delivery construct (SEQ ID NO: 5) at1, 3 and 10 mg. Data are expressed as mean±SEM, n per IL-10 deliveryconstruct dose: predose (2), 15 min (3), 30 min (3) and 45 min (3),statistical analysis not performed.

FIG. 75 illustrates tissue concentration of IL-6 measured over 24 h byimmunoassay, following pan-colonic administration of IL-10 deliveryconstruct (SEQ ID NO: 5) at 1, 3 and 10 mg. Data are expressed asmean±SEM; n per IL-10 delivery construct dose: predose (2), 15 min (3),30 min (3), 45 min (3), 8 h (1) and 24 h (1); statistical analysis notperformed.

FIG. 76 illustrates the regulation of colonic anti-inflammatory genesassessed at 8 h following pan-colonic administration of IL-10 deliveryconstruct (SEQ ID NO: 5) at 1, 3, and 10 mg doses; n per group: predose(4), all doses of IL-10 delivery construct (2). Data are expressed asmean; statistical analysis not performed. For each dose, bars from leftto right illustrate fold change observed for: CD163, SCNN1G, STC1, HGF,SGK1, MIR24-2, SCNN1B, PTGDR, MTNR1A, ACE2, NOX1, BEST2, VNN2, LTB4R2,B3GALT5, AQP8, SLC9A3, and CYP1A1.

FIG. 77 illustrates regulation of colonic pro-inflammatory genesassessed at 8 h following pan-colonic administration of IL-10 deliveryconstruct (SEQ ID NO: 5) at 1, 3, and 10 mg doses; n per group: predose(4), all doses of IL-10 delivery construct (2). Data are expressed asmean; statistical analysis not performed. For each dose, bars from leftto right illustrate: MHC-II, HPGDS, FCER1A, PLA2G2D, CCL13, FUT3, CCL28,UGT1A1, CCL20, NLRP1, and TPH.

FIG. 78 illustrates regulation of colonic pro-inflammatory genesassessed at 8 h following pan-colonic administration of IL-10 deliveryconstruct (SEQ ID NO: 5) at 1, 3, and 10 mg doses; n per group: predose(4), all doses of IL-10 delivery construct (2). Data are expressed asmean of 2-3 probes per target; statistical analysis not performed. Foreach dose, bars from left to right illustrate: MMP19, LIPG, MMP1,CHI3L1, MMP3, LAMC2, S100A8, CXCL1, FIGF, PCSK1, CASP5, CXCL2, and CHGB.

FIG. 79 illustrates regulation of colonic tissue repair genes assessedat 8 h following pan-colonic administration of IL-10 delivery construct(SEQ ID NO: 5) at 1, 3, and 10 mg doses; n per group: predose (4), alldoses of IL-10 delivery construct (2). Data are expressed as mean;statistical analysis not performed. For each dose, bars from left toright illustrate: SCNN1G, STC1, TIMP1, SCNN1B, BEST2, B3GALT5, AQP8, andSLC9A3.

FIG. 80 illustrates regulation of colonic anti-microbial genes assessedat 8 h following pan-colonic administration of IL-10 delivery construct(SEQ ID NO: 5) at 1, 3, and 10 mg doses; n per group: predose (4), alldoses of IL-10 delivery construct (2). Data are expressed as mean;statistical analysis not performed. For each dose, bars from left toright illustrate: PI15, PI3, BDKRB1, CCI28, and SERPINE2.

FIGS. 81A-81C illustrate hematoxylin and eosin staining of sections ofmouse colon. FIG. 81A is a section of naive colon, FIG. 81B is a sectionfrom a colon of a mouse treated with oxazolone, and FIG. 81C is asection of a colon from a mouse treated with oxazolone and 8.5 mg/kg ofan IL-10 delivery construct (SEQ ID NO: 5).

FIGS. 82A-82G illustrate expression of inflammatory markers upontreatment with an oral IL-10 delivery construct (SEQ ID NO: 5). FIG. 82Ashows expression of IL-4. FIG. 82B shows expression of IL-6. FIG. 82Cshows expression of IL-10. FIG. 82D shows expression of IL-17A. FIG. 82Eshows expression of IL-10. FIG. 82F shows expression of MIP1α. FIG. 82Gshows expression of GCSF/CSF3. *p<0.05; 1-way ANOVA with Tukey's posttest.

FIG. 83A illustrates the ratio of pSTAT3 to total STAT3 after treatmentwith an equimolar amount of either an IL-10 delivery construct (SEQ IDNO: 5; 1 mg/kg) or recombinant human IL-10 (0.9 mg/kg).

FIG. 83B illustrates the level of systemic IL-1Ra after treatment withan equimolar amount of either an IL-10 delivery construct (SEQ ID NO: 5;10 mg/kg) or recombinant human IL-10 (3 mg/kg).

FIG. 84 illustrates systemic expression of IL-1Ra upon treatment withthe IL-10 delivery construct (doses are shown on x axis in mg/kg).

FIG. 85A illustrates colon expression of IL-1Ra in mice treated withvehicle or 9 mg/kg of an IL-10 delivery construct (SEQ ID NO: 5) asmeasured by qPCR and normalized to the expression level in a naïvemouse.

FIG. 85B illustrates colon expression of IL-10 in mice treated withvehicle or 9 mg/kg of an IL-10 delivery construct (SEQ ID NO: 5) asmeasured by qPCR and normalized to the expression level in a naïvemouse.

FIG. 85C illustrates the ratio of IL-1Ra to IL-10 in FIGS. 85A and B.

FIG. 86 illustrates the effect of treatment with an IL-10 deliveryconstruct on the ratio of phosphorylated STAT3 (pSTAT3) to total STAT3in colon tissue.

FIG. 87 illustrates expression of pro-inflammatory markers in Macacafascicularis monkeys (about 5 to about 8 kg) administered an IL-10delivery construct by colonic sigmoidoscopy at the indicated doses.

FIG. 88 illustrates expression of anti-inflammatory markers in Macacafascicularis monkeys (about 5 to about 8 kg) administered an IL-10delivery construct by colonic sigmoidoscopy at the indicated doses.

FIG. 89 illustrates expression of biomarkers associated with tissuerepair and wound healing in Macaca fascicularis monkeys (about 5 toabout 8 kg) administered an IL-10 delivery construct by colonicsigmoidoscopy at the indicated doses.

FIGS. 90A-90B illustrate PK and PD measurements in non-human primatesfollowing administration of an IL-10 delivery construct (SEQ ID NO: 5).FIG. 90A illustrates systemic concentrations of IL-10 after delivery ofthe IL-10 delivery construct orally (PO, N=6), subcutaneously (SC, N=3),or intravenously (IV, N=3) at the indicated doses in Macaca fascicularismonkeys. FIG. 90B illustrates systemic concentrations of IL-1Ra afterdelivery of the IL-10 delivery construct orally (PO, N=6),subcutaneously (SC, N=3), or intravenously (IV, N=3) at the indicateddoses in Macaca fascicularis monkeys.

FIG. 91 illustrates the ratio of IL-1Ra to IL-10 (Ratio of the averageAUC) after delivery of an IL-10 delivery construct orally (PO, N=6),subcutaneously (SC, N=3), or intravenously (IV, N=3) at the indicateddoses in Macaca fascicularis monkeys.

FIG. 92 illustrates the turbidity of solutions comprising an IL-10delivery construct before and after vortexing, both with and without asurfactant.

FIG. 93 illustrates an SEC-HPLC chromatogram prior to vortexing.

FIG. 94 illustrates an SEC-HPLC chromatogram after vortexing.

FIG. 95 illustrates the stability of the IL-10 delivery construct in PBSwhen compacted with various different components. Samples werereconstituted in PBS at 0.3 mg/mL IL-10 delivery construct (SEQ ID NO:5) in Eppendorf vials, mounted on a rotisserie shaker at 37° C. for 5 h.Samples were withdrawn periodically for analysis by SEC.

FIG. 96 illustrates IL-10 delivery construct/excipient compatibility insolution.

FIG. 97 illustrates the compatibility of various lubricant excipientswith an IL-10 delivery construct.

FIG. 98 illustrates IL-10 delivery construct (SEQ ID NO: 5) dimerrelease from Eudragit-coated tablets (50/50 L30D55/FS30D) in a Type 4dissolution apparatus.

FIG. 99 illustrates IL-10 delivery construct (SEQ ID NO: 5) dimerrelease from Eudragit-coated tablets (20/80 L30D55/FS30D) in a Type 4dissolution apparatus.

FIG. 100 illustrates dissolution of HPMC-AS coated tables (Type 4apparatus). The dissolution started with 0.1 N HCL solution for 40 minsbefore the medium was switched to pH 7.0 phosphate buffer.

FIG. 101 illustrates IL-10 delivery construct (SEQ ID NO: 5) releasefrom HPMC-AS coated capsules (Type 4 apparatus).

FIG. 102 illustrates dissolution of HPMC-AS coated F3 tablets on a Type4 dissolution apparatus.

FIG. 103 illustrates dissolution of HPMC-AS coated F3 tablets on a Type2 dissolution apparatus.

FIGS. 104A-104C illustrate plasma concentration of various proteins(e.g., biomarkers) following oral or intravenous delivery of an IL-10delivery construct (SEQ ID NO:5). FIG. 104A illustrates plasmaconcentration of IL-10. FIG. 104B illustrates plasma concentration ofIL-1Ra. FIG. 104C illustrates concentration of IFN-γ.

FIGS. 105A-105D illustrate systemic or colon tissue concentration ofvarious biomarkers following pan-colonic delivery of IL-10 deliveryconstruct (SEQ ID NO: 5) in non-human primates (NHP). FIG. 105Aillustrates systemic concentration of IL-10. FIG. 105B illustratessystemic concentration of IL-1Ra. FIG. 105C illustrates theconcentration of IL-10 in colon tissue. FIG. 105D illustrates theconcentration of IL-10 delivery construct (SEQ ID NO: 5) in colontissue.

FIGS. 106A-106D illustrate rhIL-10 levels, as measured by ELISA, innormal and inflamed intestinal tissue (proximal, mid, and distal colon)and serum within 10 and 40 minutes of intraluminal injection of PBS,rhIL-10 (159 pmoles) or IL-10 delivery construct (SEQ ID NO: 5) (159pmoles). FIG. 106A illustrates rhIL-10 levels in normal intestinaltissue 10 minutes after intraluminal injection of PBS, rhIL-10 or IL-10delivery construct (SEQ ID NO: 5). FIG. 106B illustrates rhIL-10 levelsin inflamed intestinal tissue 10 minutes after intraluminal injection ofPBS, rhIL-10 or IL-10 delivery construct (SEQ ID NO: 5). FIG. 106Cillustrates rhIL-10 levels in normal intestinal tissue 40 minutes afterintraluminal injection of PBS, rhIL-10 or IL-10 delivery construct (SEQID NO: 5). FIG. 106D illustrates rhIL-10 levels in inflamed intestinaltissue 40 minutes after intraluminal injection of PBS, rhIL-10 or IL-10delivery construct (SEQ ID NO: 5).

FIG. 107 illustrates tissue localization of rhIL-10 and pSTAT3 afterintraluminal injection of IL-10 delivery construct (SEQ ID NO: 5) intothe jejunum of Balb/C mice.

FIG. 108 illustrates a time course analysis of pSTAT induction followingintraluminal injection of IL-10 delivery construct (SEQ ID NO: 5) intothe jejuum of Balb/C mice.

FIG. 109 illustrates immunofluorescence images of IL-10 deliveryconstruct (SEQ ID NO: 5) trafficking across intestinal epithelium indifferent murine models.

FIG. 110 illustrates pSTAT3 activity along the lamina propria of mouseintestine.

FIG. 111 illustrate IL-1Ra expression following a single dose of IL-10delivery construct (SEQ ID NO: 5) at 6 doses (1 mg, 3 mg, 10 mg, 30 mg,60 mg, 120 mg) or placebo.

FIG. 112 illustrates multiple ascending dose (MAD) escalation in a Phase1b trial of the IL-10 delivery construct (SEQ ID NO: 5).

FIG. 113 illustrates a reduction in FCP after only 14-days of treatmentwith the IL-10 delivery construct (SEQ ID NO: 5) in Ulcerative Colitis(UC) patients with baseline FCP>150 μg/g.

FIG. 114 illustrates a reduction in CRP in systemic circulation afteronly 14-days of treatment with the IL-10 delivery construct (SEQ ID NO:5) in UC patients with baseline CRP >5 mg/L.

FIG. 115 illustrates reduction in Geboes score over 14-days of treatmentwith the IL-10 delivery construct (SEQ ID NO: 5).

FIG. 116 depicts pre-dose (panel A) and post-treatment (panel B)histological images from a UC patient in the Phase 1b trial dosed with10 mg of the IL-10 delivery construct (SEQ ID NO: 5) in which the Geboesscore improved from a score of 15 to a score of three using a 22 pointscale, with higher scores indicating more severe disease activity.

FIGS. 117A-117C show microscopy images demonstrating transcytosis of anIL-10 across polarized gut epithelial cells in Wistar rats at varioustime points following luminal application of the delivery construct withthe sequence set forth in SEQ ID NO: 5 to rat jejunum. Greenfluorescence indicates the presence of IL-10 (via staining with ananti-IL-10 antibody). Blue fluorescence indicates DAPI staining, whichlabels DNA, and red fluorescence indicates the presence of CK-8(cytokeratin-8) with which a cholix-derived carrier can co-localize(e.g., in a supranuclear region of an epithelial cell) duringtranscytosis. White arrows #1 highlight the apical membrane of theepithelial cells, white arrows #2 highlight the basal membrane of theepithelial cells, and white arrow #3 indicates the presence of IL-10 inthe lamina propria. FIG. 117A demonstrates the extent of transcytosis ofIL-10 one minute after luminal application of the delivery constructwith the sequence set forth in SEQ ID NO: 5 to rat jejunum. FIG. 117Bdemonstrates the extent of transcytosis of IL-10 five minutes afterluminal application of the delivery construct with the sequence setforth in SEQ ID NO: 5 to rat jejunum. FIG. 117C demonstrates the extentof transcytosis of IL-10 ten minutes after luminal application of thedelivery construct with the sequence set forth in SEQ ID NO: 5 to ratjejunum.

FIG. 118 illustrates results of an intestinal model system of confluent,polarized human SMI-100 monolayers. An anti-hIL-10 western blot detectsthe extent of the extent of equimolar applications of an IL-10 deliveryconstruct (lane 2, apical, t=0 hr) or commercial hIL-10 (lane 3, apical,t=0 hr) transiting to the basal compartment of respective transwells(lane 4, IL-10 delivery construct, basal, t=2 hr and lane 5, hIL-10,basal, t=2 hr). Lanes from a single western blot were spliced togetherto facilitate comparisons and are indicated by black lines.

FIG. 119 illustrates dimerization of IL-10A and IL-10B receptorsengineered into U2OS osteosarcoma cells induced by an IL-10 deliveryconstruct or hIL-10 after 6 h.

FIG. 120 illustrates induction of STAT3 phosphorylation, relative tototal STAT3 content, in a mouse macrophage-like cell line J774.2 after20 min of stimulation. Data is representative of multiple studies withsimilar results.

FIG. 121 illustrates flow cytometry analysis of gated, live CD45+CD14+monocytes (PBMCs) obtained from healthy donors showing IL-10'ssuppressive effect on LPS-induced TNFα secretion; data of meanfluorescence intensity (MFI) as means±SEM (n=3) analyzed by 2-way ANOVAwith Dunnett's post-hoc test. .p<0.5, ..p<0.01, ″.p<0.001, ′. . .p<0.0001 when compared to 0 pM concentration values.

FIG. 122 illustrates flow cytometry analysis of gated, live CD45+CD14+monocytes (PBMCs) obtained from healthy donors showing IL-10'ssuppressive effect on LPS-induced IL-6 secretion; data of meanfluorescence intensity (MFI) as means±SEM (n=3) analyzed by 2-way ANOVAwith Dunnett's post-hoc test. .p<0.5, ..p<0.01, ″.p<0.001, ′. . .p<0.0001 when compared to 0 pM concentration values.

FIG. 123 illustrates flow cytometry analysis of gated, live CD45+CD14+monocytes (PBMCs) obtained from healthy donors showing IL-10'ssuppressive effect on LPS-induced surface expression of HLA-DR; data ofmean fluorescence intensity (MFI) as means±SEM (n=3) analyzed by 2-wayANOVA with Dunnett's post-hoc test. .p<0.5, ..p<0.01, ″.p<0.001, ′. . .p<0.0001 when compared to 0 pM concentration values.

FIG. 124 illustrates the oxazolone-induced colitis in BALB/c mice orallygavaged with PBS. Single channel images were captured and merged into acomposite with nuclei (blue), IL-10 (green), and pSTAT3 (red).

FIG. 125 illustrates the oxazolone-induced colitis in BALB/c mice orallygavaged with hIL-10. Single channel images were captured and merged intoa composite with nuclei (blue), IL-10 (green), and pSTAT3 (red).

FIG. 126 illustrates the oxazolone-induced colitis in BALB/c mice orallygavaged with an IL-10 delivery construct. Single channel images werecaptured and merged into a composite with nuclei (blue), IL-10 (green),and pSTAT3 (red).

FIG. 127 illustrates the percent of cells expressing pSTAT3 in smallintestine tissue segmentation.

FIG. 128 illustrates results of an hIL-10 ELISA run with PBS, IL-10, andan IL-10 delivery construct post intraluminal injection on the indicatedintestinal tissues and serum in the inflamed T cell transfer model.

FIG. 129 illustrates co-localization of the cholix derived carrier (red)and hIL-10 (green) elements of the IL-10 delivery constructdemonstrating their simultaneous transport and retention within cells ofthe lamina propria. Immunofluorescence microscopy images of rat jejunumwere obtained following a 50 uL intraluminal injection of an IL-10delivery construct prepared in PBS at ˜40 uM. For FIGS. 129-133:arrow=apical (luminal) epithelial membrane; dashed line=epithelialcell-basement membrane demarcation; l-p=lamina propria; G=goblet cell.Cell nuclei stained with DAPI (blue).

FIG. 130 illustrates staining of the hIL-10 (green) element of the IL-10delivery construct and Rab7 (red) demonstrated apical preferences forthe former and basal preferences for the latter.

FIG. 131 illustrates staining of the hIL-10 (green) element of the IL-10delivery construct and Rab11 (red) demonstrated apical preferences forthe former and basal preferences for the latter.

FIG. 132 illustrates LMAN1 reorganization and co-localization with theIL-10 delivery construct within enterocytes but not within cells oflamina propria in a time course following intraluminal injection of anIL-10 delivery construct into rat jejunum.

FIG. 133 illustrates no redistribution or co-localization of LAMP1within enterocytes but extensive co-localization within cells of laminapropria in a time course following intraluminal injection of an IL-10delivery construct into rat jejunum.

FIG. 134 illustrates localization of T cells (CDCl₃+) and pSTAT3+ cellsin mouse intestinal tissue. A pSTAT3+ CDCl₃+ cell is indicated by awhite arrow.

FIG. 135 illustrates localization of macrophages (F4/80+) and pSTAT3+cells in mouse intestinal tissue. A pSTAT3+ F4/80+ cell is indicated bya yellow arrow.

FIG. 136 illustrates higher magnification of an area of FIG. 135.pSTAT3+ F4/80+ cells are indicated by yellow arrows.

FIG. 137 illustrates additional images of pSTAT3+ F4/80+ intestinalcells.

FIG. 138 illustrates an image of pSTAT3+ F4/80+ colon cells.

FIG. 139 illustrates concentration of hIL-10 in mouse serum over a timecourse following oral gavage of 10 mg/kg of an IL-10 delivery construct.

FIG. 140 illustrates concentration of hIL-10 in mouse distal smallintestinal tissue over a time course following oral gavage of 10 mg/kgof an IL-10 delivery construct.

FIG. 141 illustrates concentration of hIL-10 in mouse colonic intestinaltissue over a time course following oral gavage of 10 mg/kg of an IL-10delivery construct.

FIG. 142 illustrates concentration of IL-1Ra in mouse serum over a timecourse following oral gavage of 10 mg/kg of an IL-10 delivery construct.

FIG. 143 illustrates concentration of the IL-10 delivery construct in asnip biopsy of colonic tissue following intracolonic spray with theindicated dose of the IL-10 delivery construct.

FIG. 144 illustrates concentration of IL-10 in a snip biopsy of colonictissue following intracolonic spray with the indicated dose of the IL-10delivery construct.

FIG. 145 illustrates the serum concentration of the IL-10 deliveryconstruct following intracolonic spray with the indicated dose of theIL-10 delivery construct.

FIG. 146 illustrates the serum concentration of IL-10 followingintracolonic spray with the indicated dose of the IL-10 deliveryconstruct.

FIG. 147 illustrates the serum concentration of IL-1Ra followingintracolonic spray with the indicated dose of the IL-10 deliveryconstruct.

FIG. 148 illustrates the ratio of pSTAT3 relative to total STAT3following intracolonic spray with the indicated dose of the IL-10delivery construct.

FIG. 149 illustrates a western blot probed for the human IL-10 (hIL-10)component of the IL-10 delivery construct of SEQ ID NO. 5 (lane 2) andcommercial rhIL-10 showing monomeric (arrow) and dimeric (double arrow)forms (lane 3). Lane 1 contains molecular weight standards.

FIG. 150 illustrates the results of reversed-phase chromatographyfollowed by mass spectrometry on the IL-10 delivery construct of SEQ IDNO. 5. Masses corresponding to both the dimer and monomer forms wereobserved.

DETAILED DESCRIPTION OF THE DISCLOSURE

IL-10 is an anti-inflammatory cytokine which can limit the damage totissues caused by infections or inflammation, making IL-10 an attractiveprotein for therapeutic drug development. A fusion protein comprisingIL-10 and a carrier, referred to herein as an IL-10 delivery construct,can be formulated into a form suitable for oral administration, such asa tablet or a capsule. Further, these tablets or capsules can beformulated in such a way as to substantially maintain the structuralintegrity of the IL-10 delivery construct dimers. Additionally, entericcoatings around these oral formulations can contribute to a distinctdissolution profile of the IL-10 delivery construct. Administration toan individual of such oral formulations can be characterized by adistinct pharmacodynamic (PD) and pharmacokinetic (PK) response in theindividual.

IL-10 is considered a master regulator of the innate and adaptive immunesystem, as it is thought to inhibit not only the inflammasome but alsomany inflammatory events found to be associated with disease includingmacrophage activation and secretion of IL-1, IL-6, TNF alpha, MMP-1/2while reducing systemic signs of inflammation and development of Tregulatory cells. There is a need for combination therapies that can beused with TNF alpha inhibitors. Providing an IL-10 delivery construct inaddition to the TNF alpha inhibitor may be efficacious and achievebetter patient outcomes.

The below terms are discussed to illustrate meanings of the terms asused in this specification, in addition to the understanding of theseterms by those of skill in the art. As used herein and in the appendedclaims, the singular forms “a,” “an,” and, “the” include pluralreferents unless the context clearly dictates otherwise. It is furthernoted that the claims can be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely,” “only,” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

Certain ranges or numbers are presented herein with numerical valuesbeing preceded by the term “about.” The term “about” is used herein tomean plus or minus 1%, 2%, 3%, 4%, or 5% of the number that the termrefers to. As used herein, the terms “subject” and “individual,” areused interchangeably and can be any animal, including mammals (e.g., ahuman or non-human animal).

As used herein, the terms “treat,” “treating” or “treatment,” and othergrammatical equivalents, include alleviating, abating or amelioratingone or more symptoms of a disease or condition, ameliorating, preventingor reducing the appearance, severity or frequency of one or moreadditional symptoms of a disease or condition, ameliorating orpreventing the underlying causes of one or more symptoms of a disease orcondition, inhibiting the disease or condition, such as, for example,arresting the development of the disease or condition, relieving thedisease or condition, causing regression of the disease or condition,relieving a condition caused by the disease or condition, or inhibitingthe symptoms of the disease or condition either prophylactically and/ortherapeutically.

As described herein, the term “percent (%) sequence identity,” and termsrelated thereto, in the context of amino acid sequences or nucleic acidsequences, is the percentage of amino acid residues or nucleic acidresidues in a candidate sequence that are identical with the amino acidresidues or nucleic acid residues, respectively, in a selected sequence,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity orpercent nucleic acid identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as Clustal Omega, BLAST, BLAST-2, ALIGN, ALIGN-2or Megalign (DNASTAR) software, with BLAST being the alignment algorithmof preference. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full-length of the sequences beingcompared, although for simplicity it may be preferred to use defaultparameters.

Interleukin-10 (IL-10) and IL-10 Delivery Constructs

The present disclosure contemplates compositions and methods fordelivery of IL-10 to a subject. As previously described, IL-10 is ananti-inflammatory cytokine which can limit the damage to tissues causedby infections or inflammation, making IL-10 an attractive protein fortherapeutic drug development. Human IL-10 exists in solution primarilyas a homodimer, where two subunits of IL-10 are non-covalentlyassociated and each subunit contains two intrachain disulfide bonds.Disruption of the dimer structure, such as by reduction or sulfitolysisof these disulfide bonds, can cause the subunits to dissociate toproduce monomers of IL-10 or aggregates thereof, which can lack thebiological activity of the dimers. Biological activity associated withIL-10 in a dimer form can comprise induction of pro-inflammatorycytokines, such as, tumor necrosis factor alpha (TNFα), interleukin-1β(IL-1β), interleukin-12 (IL-12), and interleukin-6 (IL-6). Biologicalactivity associated with IL-10 in a dimer form can comprisedownregulation of the expression of Th1 cytokines, MHC class IIantigens, and co-stimulatory molecules on macrophages; enhancing B cellsurvival, proliferation, and antibody production; blocking of NF-κBactivity; and regulating the JAK-STAT pathway.

Contemplated herein are formulations comprising IL-10, in which a highdegree of the IL-10 is maintained in dimer form. Further contemplatedherein are refolding solutions and methods for improved refoldingefficiency of IL-10-containing constructs, as well as subsequentpurification methods to further produce high levels of dimer that can bepresent in a dry (e.g., lyophilized) drug substance as well as a finaloral formulation. EXAMPLE 3 contains an exemplary refolding protocol.EXAMPLE 4 contains an exemplary purification protocol. EXAMPLE 5contains an exemplary lyophilization protocol and resulting dimercontent of pre- and post-lyophilized compositions.

In some embodiments, an IL-10 molecule is coupled to a carrier that candeliver the IL-10 across a gut epithelial cell, or a polarizedepithelial cell. This is referred to as an IL-10 delivery construct.Preferably, the IL-10 that is coupled to the carrier is in a dimer form.In some instance, the dimer is a homodimer. In some instances, the dimeris a heterodimer. In some instances, the heterodimer may comprise afirst IL-10 monomer and a variant IL-10 monomer that differs in sequencefrom the first IL-10 monomer to form a dimeric IL-10. When IL-10 is in adimer form, either a single monomer or both monomers can be coupled to acarrier. In one embodiment, each IL-10 is independently coupled to acarrier. An IL-10 delivery construct dimer can be illustrated by FIG. 1.The IL-10 delivery construct homodimer 100 can comprise two IL-10delivery constructs (e.g., SEQ ID NO: 5), each delivery constructcomprising an IL-10 101 connected by a spacer 102 to a carrier. Thecarrier can comprise a binding domain 103 and a translocation domain104.

The percent dimer in a composition can describe the percentage of thetotal number of IL-10 delivery constructs in a dimer. For example, wherea composition has three copies of an IL-10/carrier fusion protein, twoof which form a dimer, 67% of the delivery constructs can be consideredto be in dimer form.

IL-10 can be a human IL-10. Human IL-10 can comprise, consistessentially of, or consist of an amino acid sequence of SEQ ID NO: 1 orSEQ ID NO: 2. Variants of IL-10 include those having one or more aminoacid substitutions, additions and/or deletions as compared to areference sequence. Variants of IL-10 may retain the ability toupregulate IL-1Ra in colonic tissue or serum after administration byintracolonic spray in cynomolgus monkeys. In some instances, variants ofIL-10 or SEQ ID NO: 1 or SEQ ID NO: 2 are contemplated in thecompositions and methods described herein. Variants of IL-10 or SEQ IDNO: 1 or SEQ ID NO: 2 can be an amino acid sequence having at least 80%,85%, 90%, 95%, 98% or 99%% sequence identity thereto or a fragmentthereof. Variants of IL-10 may comprise amino acid substitutions at oneor more of N36, N36, D73, 187, N110, N115, K117, R128, F129, and N172relative to SEQ ID NO: 1. Variants of IL-10 may comprise one or moreamino acid substitutions such as N36Y, N36I, D73V, 187M, N110I, N115K,K117N, R128W, F129L, and N172H relative to SEQ ID NO: 1. In some cases,an IL-10 variant may comprise N36Y, N110I, K117N, and N172Hsubstitutions. In some cases, an IL-10 variant may comprise N36Y, D73V,I87M, N110I, N115K, and R128W relative to SEQ ID NO: 1. In some cases,an IL-10 variant may comprise N36I, N110I, K117N, and F129L relative toSEQ ID NO: 1.

A carrier can be a protein or another type of molecule capable oftransporting the heterologous payload across or into an epithelium(e.g., a polarized gut epithelium of a subject, such as a human). Suchtransport can include transcytosis. The transcytosis process may involveinteraction(s) of the carrier with one or more receptor(s) and/orprotein(s) on the apical and/or basal surface(s) as well as inside acell of the epithelium (e.g., a polarized gut epithelial cell). Thecarrier can be capable of transporting a heterologous payload, suchIL-10, across an epithelium without impairing the epithelium, thecarrier, and/or the biological and/or therapeutic function of thepayload.

In some embodiments, a carrier herein utilizes an endogenous traffickingpathway to transport a heterologous payload coupled thereto across apolarized epithelial cell. Such carrier can be referred to herein as atranscytosing carrier. In some instances, a carrier herein can utilizean endogenous trafficking pathway to transport a heterologous payloadcoupled thereto into a polarized epithelial cell. Such carrier can bereferred to herein as an endocytosing carrier. Within endocytosingcarriers, there can be carriers that deliver a payload coupled theretointo specific regions within the polarized epithelial cells such as anapical compartment, a supranuclear compartment, or a basal compartment

Any of the carriers herein can transport molecules coupled thereto byinteracting and/or co-localizing with one or more endogenous proteins ofsuch epithelium. The one or more endogenous proteins can be receptors orenzymes capable of moving a carrier into or across the epithelial cell.Interacting and/or co-localizing with the one or more endogenousproteins of the epithelial cell can provide a carrier with one or morefunctions, including endocytosis into the epithelial cell, avoidance ofa lysosomal destruction pathway, trafficking from an apical compartmentto a basal compartment, and/or exocytosis from the basal membrane of theepithelial cell into a submucosal compartment such as the laminapropria.

A carrier may be derived from a polypeptide secreted by a bacterium.Such a carrier may be derived from a polypeptide secreted from Vibriocholerae or Pseudomonas aeruginosa. In some embodiments, the carrier isa cholix polypeptide. In some embodiments, the carrier is a cholixpolypeptide secreted by Vibrio cholerae, while in other embodiments thecholix polypeptide is variant thereof or is derived from some otherspecies. The cholix polypeptide (e.g., a cholix polypeptide secretedfrom Vibrio cholerae or a variant thereof) can, for example, comprise asequence of any one of SEQ ID NOS: 20-146 of TABLE 2. TABLE 4illustrates exemplary carriers by identifying various amino acid residuesequences of such carriers and C-terminal positions that SEQ ID NOs20-147 can be truncated at. In some embodiments, the cholix polypeptidedoes not comprise or consist of SEQ ID NO: 126. A cholix polypeptide caninclude naturally and non-naturally occurring cholix polypeptidesequences, as well as those sequences that have at least about 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% sequence identity to a naturally (e.g.,SEQ ID NOS: 20-78 or 130-146) or non-naturally (e.g., SEQ ID NO: 3 or11) occurring cholix polypeptide described herein. A cholix polypeptidecan also include endocytosing and/or transcytosing fragments (e.g., N-and/or C-terminal truncations of cholix polypeptide) of naturally ornon-naturally occurring cholix polypeptide sequences, wherein suchendocytosing and/or transcytosing fragments can have at least about 75%,80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any of suchnaturally or non-naturally occurring cholix polypeptide sequences.

TABLE 3 provides a consensus sequence (SEQ ID NO: 147, FORMULA I) ofcholix derived polypeptides that can be used as carriers herein.

For example, a non-naturally occurring cholix polypeptide can include orconsist of the amino acid sequence set forth in SEQ ID NO: 3 or SEQ IDNO: 11 (TABLE 1). A cholix polypeptide carrier can be a truncated and/ormutated variant of a full-length cholix polypeptide. Examples oftranscytosing carriers can include those having a C-terminal truncationof any one of SEQ ID NOs 3, 11, 20-78, or 130-146, wherein theC-terminal truncation can occur at the C-terminus of the polypeptide atany amino acid position after the C-terminal residue at position 195(e.g., truncation at any one of positions 195-634 of SEQ ID NOs: 3 or11). Amino acid positions for truncation can be determined usingsequence alignment to consensus sequence SEQ ID NO: 147 or any ofreference sequences SEQ ID NO: 3 or 11. TABLE 4 below illustrates aminoacid ranges that are included in exemplary carriers and identifiesvarious C-terminal positions at which SEQ ID NOs 3, 11, 20-78 or 130-146can be truncated. In some instances, transcytosing carriers includethose having a C-terminal truncation of any of SEQ ID NOs 3, 11, 20-78,or 130-146.

A carrier can be a truncated version of a longer cholix polypeptide thatis not naturally occurring. For example, the carrier can have an aminoacids sequences that comprises or consists of amino acid residues 1-206,1-245, 1-251, 1-266, and 1-386 of SEQ ID NO: 3 or SEQ ID NO: 11.Mutation(s) in the non-naturally occurring variant can include one ormore substitution(s), deletion(s), and/or addition(s) relative to anaturally occurring cholix polypeptide. In some embodiments, a carrierherein can comprise a V1L substitution. Stated differently, in someembodiments, the cholix-related carrier has a leucine amino acid atposition “1.” (Position 1 generally refers to the first amino acid ofvariants that do not have an N-terminal methionine or the secondposition in variants that include an N-terminal methionine. In otherwords, in determining the length of a carrier, an N-terminal methionine,if present, can be ignored.) In some embodiments, carriers comprisingthe V1L substitution experience reduced or eliminated cleavage of theN-terminal amino acid. In some embodiments, carriers comprising the V1Lsubstitution experience reduced or eliminated acetylation of theN-terminal amino acid. A carrier provided herein can have a reduced(e.g., at least 50% reduced) or ablated ADP ribosylation activity (e.g.,ribosylation of elongation factor 2) relative to a naturally-occurringcholix variant. In some embodiments, the carrier can comprise anN-terminal methionine. In other embodiments, no N-terminal methionine ispresent.

A carrier herein can have a reduced (e.g., at least 50% reduced) orablated ADP ribosylation activity (e.g., ribosylation of elongationfactor 2). A carrier can be a polypeptide derived from cholix or avariant thereof that is further truncated at any one of positions 206 to633 as compared to a reference sequence, for example SEQ ID NO: 3 or SEQID NO: 11. A truncation of a cholix protein (e.g. a truncation of SEQ IDNO: 147 or variant thereof) that has the ability to transport aheterologous payload via transcytosis, such as the IL-10 deliveryconstruct, can be referred to as a functional fragment. Carriers alsoinclude variants of any of the above having at least 80%, 85%, 90%, 95%,98%, or 99% sequence identity to any of the carrier sequences herein. Inone instance, a carrier comprises SEQ ID NO: 3. In another instance, acarrier comprises SEQ ID NO: 4. Any of the carriers herein can have aV1L substitution, alone or in combination with an N-terminal methionine.In one instance, a carrier comprises SEQ ID NO: 11. In one instance, acarrier comprises SEQ ID NO: 12. Carriers also include variants of anyof the above having at least 80%, 85%, 90%, 95%, 98%, or 99% sequenceidentity to any of the sequences herein.

A carrier can be coupled to the IL-10 covalently or non-covalently,directly or indirectly. When an IL-10 is coupled to a carriercovalently, it may be coupled to the carrier directly or via a spacer.The IL-10 can be coupled to the C-terminus or the N-terminus of thecarrier. When a spacer is used to couple the IL-10 to the carrier, aspacer can include one or more amino acids. Examples of spacerscontemplated herein include oligopeptide sequences such as S, (GS)_(x),(GGS)_(x), (GGGS)_(x), (SEQ ID NO: 7) (GGGGS)_(x) (SEQ ID NO: 8), or(GGGGGS)_(x) (SEQ ID NO: 9), wherein x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15. In some cases, a spacer does not include an Sresidue adjacent to the IL-10 sequence, e.g., SEQ ID NO: 6(GGGGSGGGGSGGGG).

The carrier and/or the IL-10 can further comprise one or moremodifications on their N-terminus and/or C-terminus. Such modificationscan include an N-terminal methionine residue or other known residue foran expression in a heterologous system.

The IL-10 delivery construct can co-localize with a cell in the laminapropria expressing CD3. The cell expressing CD3 can be a lymphocyte. Thelymphocyte can be a T cell. In some embodiments, the IL-10 deliveryconstruct does not co-localize with a cell in the lamina propriaexpressing CD11c (e.g. dendritic cells), CD19 (e.g. B-lymphocytes), orCD34 (e.g. endothelia). The IL-10 delivery construct can co-localizewith a macrophage in the lamina propria. Co-localization of the IL-10delivery construct with a cell can comprise interaction or binding ofthe IL-10 delivery construct with a receptor on the surface of the cell.The carrier or the IL-10 of the IL-10 delivery construct can interact orbind with the receptor.

In some embodiments, the IL-10 delivery construct comprises, consistsessentially of, or consists of the amino acid sequence of SEQ ID NO: 5.The IL-10 delivery construct can have at least 80%, 85%, 90%, 92%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acidsequence set forth in SEQ ID NO: 5. In some embodiments, the IL-10delivery construct comprises, consists essentially of, or consists ofthe amino acid sequence of SEQ ID NO: 13. The IL-10 delivery constructcan have at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to an amino acid sequence set forth in SEQ ID NO:13. Expression and purification of IL-10, optionally with one or morecarrier and one or more spacers, as provided herein, can result in asubstantially increased concentration of a dimerized IL-10 deliveryconstruct. In one example, expression and purification of SEQ ID NO: 5can result in a composition comprising greater than 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% of the IL-10 is in a dimer form. In another example,expression and purification of SEQ ID NO: 13 can result in a compositioncomprising greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the IL-10 isin a dimer form. Such IL-10 can be a M-hIL10 or M-cholix³⁸⁶-IL-10,wherein the hIL-10 or cholix³⁸⁶-IL-10 comprises an N-terminal methionine(M). Alternatively, from 85% to 90%, from 85% to 92%, or from 85% to 95%of the IL-10 is in a dimer form.

In some embodiments, from 2% to 5% of the IL-10 is in an aggregate form.In some embodiments, no more than 2%, 3%, 4%, or 5% of the IL-10 is inan aggregate form. In some embodiments, from 5% to 7% or 6% to 7% of theIL-10 is in a monomer form. In some embodiments, no more than 5%, 6%,7%, or 8% of the IL-10 is in a monomer form.

Size exclusion chromatography (SE-HPLC) can be used to characterize thesize distribution of the IL-10 delivery construct. The percentage ofIL-10 delivery construct found in dimer, monomer, and aggregate forms ina liquid composition or a lyophilized composition if reconstituted in aliquid can be determined by SEC-HPLC (FIG. 34).

Further described herein, are non-naturally occurring nucleic acidscomprising, consisting essentially of, or consisting of a nucleic acidsequence set forth in SEQ ID NO:10, or a nucleic acid sequence at least90%, at least 92%, at least 95%, at least 98%, or at least 99% sequenceidentity to SEQ ID NO: 10. The nucleic acid can be codon optimized. Thenucleic acid can be encoded by a vector. The vector can be a plasmid ora viral vector. The viral vector can be a lentivirus, an adenovirus, anadeno-associated virus (AAV), a retrovirus, or a herpes simplex virus.The vector can be replication competent or a replication incompetent.The vector can be an integrating vector or a non-integrating vector. Acell can be transformed with any of the vectors described herein. Thecell can be a bacterial cell. The bacterial cell can be an Escherichiacoli cell. The cell can be a yeast cell. The yeast cell can be aSaccharomyces cerevisiae cell.

TABLE 1 Sequences SEQ ID NO: Description Sequence SEQ ID NO: 1 IL-10MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDI FINYIEAYMTMKIRN SEQ ID NO: 2IL-10, SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKD secretedQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAEN active formQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO: 3 Non-naturallyVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY occurringYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAP cholix variantFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTK PPYKERKDELK SEQ ID NO: 4Non-naturally VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY occurringYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAP cholix variantFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIK (cholix³⁸⁶)ISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPG LTPEQTSAGAQA SEQ ID NO: 5IL-10 delivery MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVL constructYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAGGGGSGGGGSGGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIY KAMSEFDIFINYIEAYMTMKIRNSEQ ID NO: 6 Spacer GGGGSGGGGSGGGG SEQ ID NO: DNAATGGTAGAAGAAGCGCTGAATATTTTTGACGAGTGTCGT 10 sequenceAGCCCGTGTAGCCTGACTCCGGAACCGGGCAAGCCGATT encoding IL-CAGTCCAAACTGTCTATCCCGAGCGACGTTGTGCTGGATG 10 deliveryAGGGCGTGTTGTATTACAGCATGACGATCAATGATGAGC constructAAAACGACATCAAAGACGAAGATAAGGGTGAGTCTATTATCACCATTGGCGAGTTTGCGACGGTGCGTGCAACGCGCCATTACGTGAATCAAGACGCGCCGTTTGGTGTCATTCACCTGGATATCACGACCGAAAATGGCACGAAAACCTATTCGTATAATCGCAAAGAGGGCGAGTTCGCGATCAACTGGCTGGTTCCGATCGGTGAGGATAGCCCGGCGAGCATCAAGATCAGCGTTGATGAACTGGATCAGCAGCGCAACATTATTGAAGTCCCGAAGTTGTATAGCATCGACCTGGATAATCAGACCCTGGAGCAGTGGAAAACCCAGGGTAACGTTAGCTTCTCCGTGACCCGTCCGGAGCACAACATTGCCATTAGCTGGCCGAGCGTTAGCTACAAAGCCGCACAGAAAGAGGGTTCCCGCCACAAGCGTTGGGCTCATTGGCATACCGGTTTGGCGCTGTGTTGGCTGGTGCCGATGGATGCGATCTATAACTACATCACGCAGCAAAATTGCACGCTGGGTGACAATTGGTTCGGTGGCAGCTACGAGACTGTGGCGGGTACCCCTAAGGTTATTACCGTCAAACAGGGTATTGAGCAAAAGCCTGTCGAGCAGCGTATCCACTTTAGCAAGGGTAACGCCATGTCTGCTCTGGCGGCTCATAGAGTTTGCGGCGTTCCGCTGGAGACTCTGGCCCGTTCCCGCAAGCCGCGTGACCTGACCGATGACCTGAGCTGCGCGTATCAAGCGCAAAACATTGTTAGCTTATTCGTTGCGACGCGCATTTTGTTTTCGCACCTGGATAGCGTGTTCACGCTGAACCTGGATGAACAGGAACCAGAAGTGGCAGAGCGTCTGTCAGATCTGCGTCGTATCAACGAAAACAACCCGGGCATGGTTACCCAGGTCCTTACGGTTGCACGCCAGATTTACAATGATTACGTGACCCATCACCCGGGTCTGACCCCAGAACAAACCAGCGCAGGCGCACAAGCGGGTGGCGGTGGTTCCGGTGGCGGTGGTAGCGGTGGCGGTGGTAGCCCTGGTCAAGGCACCCAATCCGAGAATAGCTGCACGCATTTTCCAGGCAATCTGCCGAATATGCTGCGTGACCTCCGCGACGCGTTCTCTCGTGTTAAGACCTTTTTTCAGATGAAAGACCAGCTGGACAATCTGCTGCTGAAAGAATCCCTGCTGGAAGATTTCAAAGGCTATCTGGGTTGCCAGGCCCTGAGCGAGATGATCCAATTCTACTTGGAAGAGGTCATGCCGCAGGCCGAAAATCAAGACCCGGACATCAAGGCACACGTGAACAGCTTGGGCGAAAACCTGAAAACCCTGCGTTTGCGCCTGCGTCGTTGTCACCGTTTCCTGCCGTGCGAGAATAAGAGCAAAGCCGTCGAACAAGTCAAAAATGCATTCAACAAGCTGCAAGAGAAAGGTATCTACAAGGCTATGAGCGAGTTTGACATTTTCATTAACTACATTG AAGCGTACATGACCATGAAGATCCGTAACSEQ ID NO: Non-naturally LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY 11occurring YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAP cholix variantFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIK (V1L)ISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTK PPYKERKDELK SEQ ID NO:Non-naturally LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY 12 occurringYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAP cholix variantFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIK (cholix³⁸⁶)ISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVT (V1L)RPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPG LTPEQTSAGAQA SEQ ID NO:IL-10 delivery MLEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVL 13 constructYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDA (cholix V1L)PFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAGGGGSGGGGSGGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIY KAMSEFDIFINYIEAYMTMKIRNSEQ ID NO: Humira Fab DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPG 151light chain KAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC SEQ ID NO: Humira FabEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQA 152 heavy chainPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCSEQ ID NO: Remicade Fab DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGS 153light chain PRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC SEQ ID NO: Remicade FabEVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQS 154 heavy chainPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYYGSTYDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKT

TABLE 2 Additional cholix polypeptides SEQ ID NO: 130VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISTKPPYKERKDELKSEQ ID NO: 131 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAKQSIAKQSIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISAKPPYKEQKDELK SEQ ID NO: 132VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIMDEGKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAKQSIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK SEQ ID NO: 133VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYERLTPAEEAVVKEAIAKEQSISAKPPYKEQKDELK SEQ ID NO: 134VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYERLTPAEEAVVKEAIAKEQSISAKPPYKEQKDELK SEQ ID NO: 135VEDELNIFDECRSPCSLTPEPGKQIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCVYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEKKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISTKPPYKERKDELKSEQ ID NO: 136 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKERKDELKSEQ ID NO: 137 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVSTHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISTKPPYKERKDELKSEQ ID NO: 138 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCVYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEKKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISTKPPYKERKDELKSEQ ID NO: 139 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIHRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISTKPPYKERKDELKSEQ ID NO: 140 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASDNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISTKPPYKERKDELKSEQ ID NO: 141 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLIPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISAKPPYKERKDELKSEQ ID NO: 142 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLIPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISAKPPYKEQKDELKSEQ ID NO: 143 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRMLFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLIPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKEQKDELKSEQ ID NO: 144 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARLKKGTGNAELPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITHVIGHSLPLRNEAFTGPERVDGEDETVIGWDMAIHAVAIPSTIPGNAYEVLAIDEEAVAEE QSISAKPPYKERKDELKSEQ ID NO: 145 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVFFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVTERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIHRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISTKPPYKERKDELKSEQ ID NO: 146 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKERKDELKSEQ ID NO: 20 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVALNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKERKDELKSEQ ID NO: 21 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQAPEVAERLSALRQINENNPGVVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGNGGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISAKPPYKEQKDELKSEQ ID NO: 22 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCVYQAQNIVSLFVATRILFSHLDSVFTLNLEEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGNGGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITDVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISAKPPYKEQKDELKSEQ ID NO: 23 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAIMVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRYLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPERVDGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISPKPPYKERKDELKSEQ ID NO: 24 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKDGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSAIRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASDNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISTKPPYKERKDELKSEQ ID NO: 25 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKDGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKHCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKERKDELKSEQ ID NO: 26 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWRTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLEEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQS ISTKPPYKERKDELKSEQ ID NO: 27 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSNGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISTKPPYKERKDELKSEQ ID NO: 28 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLLENRAVITPQGVINWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISTKPPYKERKDELKSEQ ID NO: 29 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQKNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKEQKDELKSEQ ID NO: 30 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIYAVAIPSTIPGNAYEELAIDEEAVAKEQ SISAKPPYKEQKDELKSEQ ID NO: 31 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAK EQSISAKPPYKEQKDELKSEQ ID NO: 32 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIYAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKEQKDELKSEQ ID NO: 33 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQKNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISTKPPYKERKDELKSEQ ID NO: 34 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCVYQAQNIVSLFVATRILFSHLDSVFTLNLEEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPERVDGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISTKPPYKERKDELKSEQ ID NO: 35 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGNGGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITDVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISAKPPYKEQKDELKSEQ ID NO: 36 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAIHWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCVYQAQNIVSLFVATRILFSHLDSVFTLNLEEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISTKPPYKERKDELKSEQ ID NO: 37 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATIRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKEQKDELKSEQ ID NO: 38 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITFGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAK EQSISTKPPYKERKDELKSEQ ID NO: 39 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNSDQANINIESRSGRSYLPENRAVITQQGVTNWTYQELEATHQALTQEGYVFVGYHGTNHVAAQSIVNRISPVPRGSDTESERAWGGLYVSTDASVAYGYARIQEGTADGGGLTPAERKARGVMLRVYLPQASLERFYRINADLEKERNLVERVIGHPLPLRNEAFTGTDAEEGSDETAIGWDMAIHGVAIPSTIPGNSYAQLPIDEEAVAKEQSISA KPPYKEQKDELKSEQ ID NO: 40 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISTKPPYKERKDELKSEQ ID NO: 41 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASDNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISTKPPYKERKDELKSEQ ID NO: 42 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQNIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ SISTKPPYKERKDELKSEQ ID NO: 43 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDIAIHAVAIPSTIPGNAYEELAIDEEAVAKEQS ISTKPPYKERKDELKSEQ ID NO: 44 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKERKDELKSEQ ID NO: 45 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMETLAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKERKDELKSEQ ID NO: 46 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKEQKDELKSEQ ID NO: 47 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMETLAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKEQKDELKSEQ ID NO: 48 VEDELNIFDECRSPCLLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKEQKDELKSEQ ID NO: 49 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVIPGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMETLAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKE QSISAKPPYKERKDELKSEQ ID NO: 50 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEGLTTDEEAVVKEAIAKEQS ISAKPPYKERKDELKSEQ ID NO: 51 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSQKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEGLTTDEEAVVKEAIAKE QSISAKPPYKEQKDELKSEQ ID NO: 52 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEGLTTDEEAVVKEAIAKEQS ISAKPPYKERKDELKSEQ ID NO: 53 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEGLTTDEEAVVKEAIAKEQS ISAKPPYKEQKDELKSEQ ID NO: 54 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEGLTTDEEAVVKEAIAKEQS ISAKPPYKEQKDELKSEQ ID NO: 55 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGIPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEGLTTDEEAVVKEAIAKEQS ISAKPPYKERKDELKSEQ ID NO: 56 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGIPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEGLTTDEEAVVKEAIAKEQS ISAKPPYKEQKDELKSEQ ID NO: 57 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPEVVLCFFEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNSDQANINIESRSGRSYLPENRAVITQQGVTNWTYQELEATHQALTQEGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEGLTTDEEAVVKEAIAKEQSI SAKPPYKEQKDELKSEQ ID NO: 58 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSFNRKEGEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAQKDGARHKRWAHWHTGLALCWLVPLDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGMEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARGRKPRDLTDDLQCAYQAQNIVSLFLATRILFSHLDSVFTLNLDEQEPEVAERLTDLRRINENNPGMVTQVLTIARQIYNDYVTEHPGLTPEQTSAGAQAADILSLFCPDADESCVASNSDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEAKHQTLTREGYVFVGYHGTNHVAAQSIVNRITPVPRGNNTEKEEEWGGVYVATHAELAHRYARIKEGTGENGLPTTEEKKSRGVMLRVYLPRASLERFYRTNIPLENADEHVTQVIGHPLPLRNEAFTGPESAGGEDETAIGWDMAIHGVAIPSTIPGNSYAQLPIDEEAVAKE QSISAKPPYKEHDELKSEQ ID NO: 59 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSFNRKEGEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAQKDGARHKRWAHWHTGLALCWLVPLDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGMEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARGRKPRDLTDDLQCAYQAQNIVSLFLATRILFSHLDSVFTLNLDEQEPEVAERLTDLRRINENNPGMVTQVLTIARQIYNDYVTEHPGLTPEQTSAGAQAADILSLFCPDADESCVASNSDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEAKHQTLTREGYVFVGYHGTNHVAAQSIVNRITPVPRGNNTEKEEEWGGVYVATHAEVNHRYARIKEGTGENGLPTTEEKKSRGVMLRVYLPRASLERFYRTNIPLENADEHVTQVIGHPLPLRNEAFTGPESAGGEDETAIGWDMAIHGVAIPSTIPGNSYAQLPIDEEAVAKE QSISAKPPYKEHDELKSEQ ID NO: 60 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSFNRKEGEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAQKDGARHKRWAHWHTGLALCWLVPLDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGMEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARGRKPRDLTDDLQCAYQAQNIVSLFLATRILFSHLDSVFTLNLDEQEPEVAERLTDLRRINENNPGMVTQVLTIARQIYNDYVTEHPGLTPEQTSAGAQAADILSLFCPDADESCVASNSDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEAKHQTLTREGYVFVGYHGTNHVAAQSIVNRITPVPRGNNTEKEEEWGGVYVATHAELAHRYARIKEGTGENGLPTTEKKKSRGVMLKVYLPRASLERFYRTNIPLENADEHVTQVIGHPLPLRNEAFTGPESAGGENETAIGWDMAIHGVAIPSTIPGNSYAQLPIDEEAVAK EQSISAKPPYKEHDELKSEQ ID NO: 61 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEGLTTDEEAVVKEAIAKEQS ISAKPPYKEQKDELKSEQ ID NO: 62 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVIPGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEGLTTDEEAVVKEAIAKEQS ISAKPPYKERKDELKSEQ ID NO: 63 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSFNRKEGEFAINWLVIPGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAQKDGARHKRWAHWHTGLALCWLVPLDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGMEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARGRKPRDLTDDLQCAYQAQNIVSLFLATRILFSHLDSVFTLNLDEQEPEVAERLTDLRRINENNPGMVTQVLTIARQIYNDYVTEHPGLTPEQTSAGAQAADILSLFCPDADESCVASNSDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEAKHQTLTREGYVFVGYHGTNHVAAQSIVNRITPVPRGNNTEKEEEWGGVYVATHAELAHRYARIKEGTGENGLPTTEEKKSRGVMLRVYLPRASLERFYRTNIPLENADEHVTQVIGHPLPLRNEAFTGPESAGGEDETAIGWDMAIHGVAIPSTIPGNSYAQLPIDEEAVAKE QSISAKPPYKEHDELKSEQ ID NO: 64 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPP YKERKDELKSEQ ID NO: 65 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPP YKERKDELKSEQ ID NO: 66 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEQSIAISWPSVSYNAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKP PYKERKDELKSEQ ID NO: 67 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGNGGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISAKPP YKEQKDELKSEQ ID NO: 68 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPERVDGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPP YKERKDELKSEQ ID NO: 69 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKMYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPERVDGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTK PPYKERKDELKSEQ ID NO: 70 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQKRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEEPELCTYGEDWHGGAYKTVAGTPEAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLQDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISAKPP YKEQKDELKSEQ ID NO: 71 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPEVVLCFFEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNSDQANINIESRSGRSYLPENRAVITQQGVTNWTYQELEATHQALTQEGYVFVGYHGTNHVAAQSIVNRISPVPRGSDTESERAWGGLYVSTDASVAYGYARIQEGTADGGGLTPAERKARGVMLRVYLPQASLERFYRINADLEKERNLVERVIGHPLPLRNEAFTGTDAEEGSDETAIGWDMAIHGVAIPSTIPGNSYAQLPIDEEAVAKEQSISAKPPYK EQKDELKSEQ ID NO: 72 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPEVVLCFFEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPLLTPEQTSAGAQAADILSLFCPDADKSCVASNSDQANINIESRSGRSYLPENRAVITQQGVTNWTYQELEATHQALTQEGYVFVGYHGTNHVAAQSIVNRISPVPRGSDTESERAWGGLYVSTDASVAYGYARIQEGTADGGGLTPAERKARGVMLRVYLPQASLERFYRINADLEKERNLVERVIGHPLPLRNEAFTGTDAEEGSDETAIGWDMAIHGVAIPSTIPGNSYAQLPIDEEAVAKEQSISAKPPYK EQKDELKSEQ ID NO: 73 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPEVVLCFFEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNSDQANINIESRSGRSYLPENRAVITQQGVTNWTYQELEATHQALTQEGYVFVGYHGTNHVAAQSIVNRISPVPRGSDTESERAWGGLYVSTDASVAYGYARIQEGTADGGGLTPAERKARGVMLRVYLPQASLERFYRINADLEKERNLVERVIGHPLPLRNEAFTGTDAEEGSDETAIGWDMAIHGVAIPSTIPGNSYAQLPIDEEAVAKEQSISAKPPYK EQKDELKSEQ ID NO: 74 VEDELNIFDECRSPCSLTPEPGKPIQSKLFIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAIEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPY KERKDELKSEQ ID NO: 75 VEDELNIFDECRSPCSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWYGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDTDKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPP YKERKDELKSEQ ID NO: 76 VEDELKIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISAKPP YKEQKDELKSEQ ID NO: 77 VEDELKIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQAPEVAERLSDLRRINEDNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELETTHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISAKPP YKEQKDELKSEQ ID NO: 78 VEDELKIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKAVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGNGGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIYAVAIPSTIPGNAYEELAIDEEAVAKEQSISAK PPYKEQKDELKSEQ ID NO: 79 TPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNSDQANINIESRSGRSYLPENRAVITQQGVTNWTYQELEATHQALTQEGYVFVGYHGTNHVAAQSIVNRISPVPRGSDTESERAWGGLYVSTDASVAYGYARIQEGTADGGGLTPAERKARGVMLRVYLPQASLERFYRINADLEKERNLVERVIGHPLPLRNEAFTGTDAEEGSDETAIGWDMAIHGVAIPSTIPGNSYAQLPIDEEAVAKEQSISAKPPYKEQKDELK SEQ ID NO: 80SIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK SEQ ID NO: 81MTINDEQNDIMDEGKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAKQSIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK SEQ ID NO: 82CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIMDEGKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFTINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAKQSIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETIIGWDMAIHAVAIPS SEQ ID NO: 83CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIMDEGKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAKQSIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPS SEQ ID NO: 84CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIMDEGKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAKQSIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPS SEQ ID NO: 85MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKAVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGNGGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIYAVAIPSTIPGNAYEELAIDEEAVAK EQSISAKPPYKEQKDELKSEQ ID NO: 86 MTINDEQNDIKDEDKGESIITIGDFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPERVDGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK SEQ ID NO: 87CSLTPEPGKPIQSQLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCVYQAQNIVSLFVATRILFSHLDSVFTLNLEEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPERAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 88 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 89 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 90 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLIPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 91 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCVYQAQNIVSLFVATRILFSHLDSVFTLNLEEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGNGGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITDVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 92 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVALNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 93 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAIMVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRYLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPERVDG EDETVIGWDMAIHAVAIPSSEQ ID NO: 94 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKDGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSAIRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASDNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 95 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKDGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASDNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYLTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGE DETVIGWDMAIHAVAIPSSEQ ID NO: 96 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 97 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYTRIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 98 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFYPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 99 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGNGGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 100 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQKNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 101 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 102 CSLTPELGKPIQSKLSISSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPS SEQ ID NO: 103CSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 104 CSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLEEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 105 CSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASDNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 106 CSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINVESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEREARGVMLRVYIPRASLERFYRTNTPLENAERHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPS SEQ ID NO: 107CSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPERVDG EDETVIGWDMAIHAVAIPSSEQ ID NO: 108 CSLTPELGKPIQSKLSIPSDVVLDEGVLYYSMTINDDQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKPCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQNIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGG EDETVIGWDMAIHAVAIPSSEQ ID NO: 109 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIMDEGKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAKQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFFEDPELCTYGEDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTLLENAEEHITQVIGHSLPLRNEAFTGPESAGGE DETVIGWDMAIHAVAIPSSEQ ID NO: 110 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIG WDMAIHAVAIPSSEQ ID NO: 111 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHAAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIG WDMAIHAVAIPSSEQ ID NO: 112 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETV IGWDMAIHAVAIPSSEQ ID NO: 113 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQAPEVAERLSDLRRINEDNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELETTHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETV IGWDMAIHAVAIPSSEQ ID NO: 114 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGNGGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETV IGWDMAIYAVAIPSSEQ ID NO: 115 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEIDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWFTTSPKVTLCFYEDPAQCTYGDDWHGGAYKTVAGIPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAEQETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIG WDMAIHAVAIPSSEQ ID NO: 116 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEEPELCTYGEDWHGGAYKTVAGTPGAITVKQGIEQKTVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIG WDMAIHAVAIPSSEQ ID NO: 117 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQKRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEEPELCTYGEDWHGGAYKTVAGTPEAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIG WDMAIHAVAIPSSEQ ID NO: 118 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRVVITPQGVTNWTYQELDATHQALTREDYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERETRGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDET VIGWDMAIHAVAIPSSEQ ID NO: 119 CSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPEVVLCFFEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNSDQANINIESRSGRSYLPENRAVITQQGVTNWTYQELEATHQALTQEGYVFVGYHGTNHVAAQSIVNRISPVPRGSDTESERAWGGLYVSTDASVAYGYARIQEGTADGGGLTPAERKARGVMLRVYLPQASLERFYRINADLEKERNLVERVIGHPLPLRNEAFTGTDAEEGSDETAIGW DMAIHGVAIPSSEQ ID NO: 120 CSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPEVVLCFFEDPELCTYGDDWHGGAYKTVAGTPKAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSASAQAADILSLFCPDADKSCVASNSDQANINIESRSGRSYLPENRAVITQQGVTNWTYQELEATHQALTQEGYVFVGYHGTNHVAAQSIVNRISPVPRGSDTESERAWGGLYVSTDASVAYGYARIQEGTADGGGLTPAERKARGVMLRVYLPQASLERFYRINADLEKERNLVERVIGHPLPLRNEAFTGTDAEEGSDETAIGW DMAIHGVAIPSSEQ ID NO: 121 VEDELNIFDECRSPCSLTPEPGKQIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVINLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLTDDLSCVYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEKKWGGLYVATHAEVAHGYARIKEGTGEY GLPTRAERDARGVMLRVSEQ ID NO: 122 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKEGEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAEKDGARHKRWAHWHTGLALCWLVPLDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGMEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARGRKPRDLTDDLQCAYQAQNIVSLFLATRILFSHLDSVFTLNLDEQEPEVAERLTDLRRINENNPGMVTQVLTIARQIYNDYVTEHPGLTPEQTSAGAQAADILSLLCPDADGSCVASNSDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEAKHQTLTREGYVFVGYHGTNHVAAQSIVNRITPVPRGNNTEKEEEWGG SEQ ID NO: 123VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSFNRKEGEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWETQGNVSFAVTRPEQSIAISWPSVSYKAAEKDGARHKRWAHWHTGLALCWLVPLDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGMEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARGRKPRDLTDDLQCAYQAQNIVSLFLATRILFSHLDSVFTLNLDEQEPEVAERLTDLRRINENNPGMVTQVLTIARQIYNDYVTEHPGLTPEQTSAGAQAADILSLFCPDADESCVASNSDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEAKHQTLTREGYVFVGYHGTNHVAAQSIVNRITPVPRGNNTEKEEEWGG SEQ ID NO: 124YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSNGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISAKPPYKERKDELK SEQ ID NO: 125VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQKRNIIEVPKLYSIDLDNQTLEQWENQGNVSFAVTRPEQSIAISWPSVSYKAAHKNGSRHKRWANWLTTLPKVVLCFYEEPELCTYGEDWHGGAYKTVAGTPEAITVKQGIEQKTVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLQDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHV SEQ ID NO: 126VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNSDQANINIES SEQ ID NO: 127LFSHLDSVFTLNLHEQEPAVAERLSALRQINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISAK PPYKERKDELKSEQ ID NO: 128 AVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKER KDELKSEQ ID NO: 129 AVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKER KDEL

TABLE 3 FORMULA I SEQ ID NO:X1-E-X3-X4-L-X6-I-F-D-E-C-R-S-P-C-X16-L-T-P-E-X21-G-K-X24-I-Q-S- 147K-L-X30-I-P-X33-D-V-V-L-D-E-G-V-L-Y-Y-S-M-T-I-N-D-E-Q-N-D-I-X56-D-E-X59-K-G-E-S-I-I-T-X67-G-E-F-A-T-X73-R-A-T-R-H-Y-V-X81-Q-D-A-P-F-G-V-I-X90-L-D-I-T-T-E-N-G-T-K-X101-Y-S-X104-N-R-K-X108-X109-E-F-X112-I-X114-W-L-V-X118-X119-G-E-D-S-P-A-S-I-K-I-S-X131-D-E-X134-D-Q-X137-R-N-I-I-E-V-P-K-L-Y-S-I-D-L-D-N-Q-T-L-E-Q-W-X160-X161-Q-G-N-V-X166-F-X168-V-T-R-P-E-X174-X175-I-A-I-S-W-P-S-V-S-Y-X186-A-A-X189-K-X191-G-X193-R-H-K-R-W-A-X200-W-X202-T-X204-X205-X206-X207-X208-X209-L-X211-X212-X213-X214-X215-X216-X217-X218-X219-X220-X221-X222-X223-X224-C-T-X227-G-X229-X230-W-X232-G-G-X235-Y-X237-T-V-A-G-X242-P-X244-X245-I-X247-V-K-Q-G-X252-E-Q-K-X256-V-E-Q-R-I-H-F-S-X265-X266-N-A-X269-X270-X271-L-A-A-H-R-V-C-G-V-P-L-E-T-L-A-R-X288-R-K-P-R-X293-L-X295-D-D-L-X299-C-X301-Y-X303-A-Q-X306-I-V-S-L-F-X312-A-T-R-X316-L-F-X319-H-X321-D-S-X324-F-T-L-N-L-X330-X331-Q-X333-P-X335-V-X337-E-R-L-X341-X342-X343-R-X345-I-N-E-X349-N-P-G-X353-V-X355-Q-V-L-T-X360-A-R-Q-I-Y-N-D-Y-V-T-X371-H-P-X374-L-X376-P-E-Q-T-S-A-X383-A-Q-A-A-D-I-L-S-L-X393-X394-P-D-X397-D-X399-X400-C-V-A-X404-X405-X406-D-Q-A-N-I-N-X413-E-S-R-S-G-R-S-Y-L-X423-E-N-R-A-V-I-T-X431-Q-G-V-T-N-W-T-Y-Q-E-L-X443-X444-X445-H-Q-X448-L-T-X451-E-X453-Y-V-F-V-G-Y-H-G-T-N-H-X465-A-A-Q-X469-I-V-N-R-I-X475-P-V-P-R-G-X481-X482-T-E-X485-E-X487-X488-W-G-G-X492-Y-V-X495-T-X497-A-X499-X500-X501-X502-X503-Y-X505-R-X507-X508-X509-G-T-X512-X513-X514-X515-X516-X517-T-X519-X520-X521-X522-X523-X524-R-G-V-M-L-X530-V-Y-X533-X534-X535-A-S-L-E-R-F-Y-R-X544-N-X546-X547-L-E-X550-X551-X552-X553-X554-X555-X556-X557-V-I-G-H-X562-L-P-L-R-N-E-A-F-T-G-X573-X574-X575-X576-X577-G-X579-X580-E-T-X583-I-G-W-D-X588-A-I-X591-X592-V-A-I-P-S-T-I-P-G-N-X603-Y-X605-X606-L-X608-X609-X610-E-E-A-X614-A-X616-E-Q-S-I-S-X622-K-P-P-Y-K-E-X629-X630-D-E-L-K;

-   -   wherein X1 is selected from the group consisting of V and L; X3        is selected from the group consisting of E and D; X4 is selected        from the group consisting of A and E; X6 is selected from the        group consisting of N and K; X16 is selected from the group        consisting of S and L; X21 is selected from the group consisting        of P and L; X24 is selected from the group consisting of P and        Q; X30 is selected from the group consisting of S and F; X33 is        selected from the group consisting of S and G; X56 is selected        from the group consisting of K and M; X59 is selected from the        group consisting of D and G; X67 is selected from the group        consisting of I and F; X73 is selected from the group consisting        of V and I; X81 is selected from the group consisting of N and        S; X90 is selected from the group consisting of H and N; X101 is        selected from the group consisting of T and M; X104 is selected        from the group consisting of Y and F; X108 is selected from the        group consisting of E and D; X109 is selected from the group        consisting of G and S; X112 is selected from the group        consisting of A and T; X114 is selected from the group        consisting of N and H; X118 is selected from the group        consisting of P and I; X119 is selected from the group        consisting of I and P; X131 is selected from the group        consisting of V and I; X134 is selected from the group        consisting L and I; X137 is selected from the group consisting Q        and K; X160 is selected from the group consisting K and E; X161        is selected from the group consisting T and N; X166 is selected        from the group consisting S and F; X168 is selected from the        group consisting S and A; X174 is selected from the group        consisting H and Q; X175 is selected from the group consisting        N, S, SIAKQS (SEQ ID NO: 148), and SIAKQSIAKQS (SEQ ID NO: 149);        X186 is selected from the group consisting of K and N; X189 is        selected from the group consisting of Q, E, and H; X191 is        selected from the group consisting of E, N, and D; X193 is        selected from the group consisting of S and A; X200 is selected        from the group consisting of H and N; X202 is selected from the        group consisting of H, L, F, and R; X204 is selected from the        group consisting of G and T; X205 is selected from the group        consisting of L and S; X206 is selected from the group        consisting of A and P; X207 is selected from the group        consisting of L, E, and K; X208 is selected from the group        consisting of C and V; X209 is selected from the group        consisting of W, V, and T; X211 is selected from the group        consisting of V and no amino acid; X212 is selected from the        group consisting of P and no amino acid; X213 is selected from        the group consisting of M, I, L, and no amino acid; X214 is        selected from the group consisting of D and no amino acid; X215        is selected from the group consisting of A and no amino acid;        X216 is selected from the group consisting of I and no amino        acid; X217 is selected from the group consisting of Y and C;        X218 is selected from the group consisting of N and F; X219 is        selected from the group consisting of Y and F; X220 is selected        from the group consisting of I and E; X221 is selected from the        group consisting of T and D; X222 is selected from the group        consisting of Q and P; X223 is selected from the group        consisting of Q, E, and A; X224 is selected from the group        consisting of N, L, and Q; X227 is selected from the group        consisting of L and Y; X229 is selected from the group        consisting of D and E; X230 is selected from the group        consisting of N and D; X232 is selected from the group        consisting of F, H, and Y; X235 is selected from the group        consisting of S and A; X237 is selected from the group        consisting of E and K; X242 is selected from the group        consisting of T and I; X244 is selected from the group        consisting of K, E, and G; X245 is selected from the group        consisting of V and A; X247 is selected from the group        consisting of T and M; X252 is selected from the group        consisting of I and M; X256 is selected from the group        consisting of P, T, and A; X265 is selected from the group        consisting of K, Q, and N; X266 is selected from the group        consisting of G and K; X269 is selected from the group        consisting of M and I; X270 is selected from the group        consisting of S and E; X271 is selected from the group        consisting of A and T; X288 is selected from the group        consisting of S and G; X293 is selected from the group        consisting of D and Y; X295 is selected from the group        consisting of T, P, and Q; X299 is selected from the group        consisting of S and Q; X301 is selected from the group        consisting of A and V; X303 is selected from the group        consisting of Q and N; X306 is selected from the group        consisting of N and Q; X312 is selected from the group        consisting of V and L; X316 is selected from the group        consisting of I and M; X319 is selected from the group        consisting of S and T; X321 is selected from the group        consisting of L and I; X324 is selected from the group        consisting of V and I; X330 is selected from the group        consisting of D, E, and H; X331 is selected from the group        consisting of E and G; X333 is selected from the group        consisting of E and A; X335 is selected from the group        consisting of E and A; X337 is selected from the group        consisting of A and T; X341 is selected from the group        consisting of S, D, and T; X342 is selected from the group        consisting of D and A; X343 is selected from the group        consisting of L and I; X345 is selected from the group        consisting of R and Q; X349 is selected from the group        consisting of N and D; X353 is selected from the group        consisting of M and V; X355 is selected from the group        consisting of T and I; X360 is selected from the group        consisting of V and I; X371 is selected from the group        consisting of H and E; X374 is selected from the group        consisting of G and L; X376 is selected from the group        consisting of T and I; X383 is selected from the group        consisting of G and S; X393 is selected from the group        consisting of F and L; X394 is selected from the group        consisting of C and Y; X397 is selected from the group        consisting of A and T; X399 is selected from the group        consisting of K, E, and G; X400 is selected from the group        consisting of S, P, and H; X404 is selected from the group        consisting of S and L; X405 is selected from the group        consisting of N and D; X406 is selected from the group        consisting of N and S; X413 is selected from the group        consisting of I and V; X423 is selected from the group        consisting of P and L; X431 is selected from the group        consisting of P and Q; X443 is selected from the group        consisting of E and D; X444 is selected from the group        consisting of A and T; X445 is selected from the group        consisting of T and K; X448 is selected from the group        consisting of A and T; X451 is selected from the group        consisting of R and Q; X453 is selected from the group        consisting of G and D; X465 is selected from the group        consisting of V and A; X469 is selected from the group        consisting of T, S, and N; X475 is selected from the group        consisting of A, S, and T; X481 is selected from the group        consisting of N and S; X482 is selected from the group        consisting of N and D; X485 is selected from the group        consisting of N, S, and K; X487 is selected from the group        consisting of E, R, and K; X488 is selected from the group        consisting of K, A, and E; X492 is selected from the group        consisting of L and V; X495 is selected from the group        consisting of A and S; X497 is selected from the group        consisting of H and D; X499 is selected from the group        consisting of E and S; X500 is selected from the group        consisting of V and L; X501 is selected from the group        consisting of A and N; X502 is selected from the group        consisting of H and Y; X503 is selected from the group        consisting of G and R; X505 is selected from the group        consisting of A and T; X507 is selected from the group        consisting of I and L; X508 is selected from the group        consisting of K and Q; X509 is selected from the group        consisting of E and K; X512 is selected from the group        consisting of G and A; X513 is selected from the group        consisting of E, D, and N; X514 is selected from the group        consisting of Y, G, A, and N; X515 is selected from the group        consisting of G and E; X516 is selected from the group        consisting of L and G; X517 is selected from the group        consisting of P and L; X519 is selected from the group        consisting of R, P, and T; X520 is selected from the group        consisting of A and E; X521 is selected from the group        consisting of E and K; X522 is selected from the group        consisting of R, Q, and K; X523 is selected from the group        consisting of D, K, and E; X524 is selected from the group        consisting of A, T, and S; X530 is selected from the group        consisting of R and K; X533 is selected from the group        consisting of I and L; X534 is selected from the group        consisting of P and H; X535 is selected from the group        consisting of R and Q; X544 is selected from the group        consisting of T and I; X546 is selected from the group        consisting of T, A, and I; X547 is selected from the group        consisting of P and D; X550 is selected from the group        consisting of N and K; X551 is selected from the group        consisting of A and E; X552 is selected from the group        consisting of E, R, and D; X553 is selected from the group        consisting of E, N, and R; X554 is selected from the group        consisting of H and L; X555 is selected from the group        consisting of I and V; X556 is selected from the group        consisting of T and E; X557 is selected from the group        consisting of Q, R, H, and D; X562 is selected from the group        consisting of S and P; X573 is selected from the group        consisting of P and T; X574 is selected from the group        consisting of E and D; X575 is selected from the group        consisting of S, A, and R; X576 is selected from the group        consisting of A, E, and V; X577 is selected from the group        consisting of G, E, and D; X579 is selected from the group        consisting of E and S; X580 is selected from the group        consisting of D and N; X583 is selected from the group        consisting of V and A; X588 is selected from the group        consisting of M and I; X591 is selected from the group        consisting of H and Y; X592 is selected from the group        consisting of A and G; X603 is selected from the group        consisting of A and S; X605 is selected from the group        consisting of E and A; X606 is selected from the group        consisting of E, A, Q, G, V, and R; X608 is selected from the        group consisting of A, P, and T; X609 is selected from the group        consisting of I, T, and P; X610 is selected from the group        consisting of D and A; X614 is selected from the group        consisting of V and VVKEAI (SEQ ID NO: 150); X616 is selected        from the group consisting of K and E; X622 is selected from the        group consisting of T, A, and P; and X629 is selected from the        group consisting of R, Q, and H; and X630 is selected from the        group consisting of K and no amino acid.

TABLE 4 Exemplary Transcytosing Carriers Identifying Amino Acid Residuesof any one of SEQ ID NOs: 20-147 AA residues 1-195 1-196 1-197 1-1981-199 1-200 1-201 1-202 1-203 1-204 1-205 1-206 1-207 1-208 1-209 1-2101-211 1-212 1-213 1-214 1-215 1-216 1-217 1-218 1-219 1-220 1-221 1-2221-223 1-224 1-225 1-226 1-227 1-228 1-229 1-230 1-231 1-232 1-233 1-2341-235 1-236 1-237 1-238 1-239 1-240 1-241 1-242 1-243 1-244 1-245 1-2461-247 1-248 1-249 1-250 1-251 1-252 1-253 1-254 1-255 1-256 1-257 1-2581-259 1-260 1-261 1-262 1-263 1-264 1-265 1-266 1-267 1-268 1-269 1-2701-271 1-272 1-273 1-274 1-275 1-276 1-277 1-278 1-279 1-280 1-281 1-2821-283 1-284 1-285 1-286 1-287 1-288 1-289 1-290 1-291 1-292 1-293 1-2941-295 1-296 1-297 1-298 1-299 1-300 1-301 1-302 1-303 1-304 1-305 1-3061-307 1-308 1-309 1-310 1-311 1-312 1-313 1-314 1-315 1-316 1-317 1-3181-319 1-320 1-321 1-322 1-323 1-324 1-325 1-326 1-327 1-328 1-329 1-3301-331 1-332 1-333 1-334 1-335 1-336 1-337 1-338 1-339 1-340 1-341 1-3421-343 1-344 1-345 1-346 1-347 1-348 1-349 1-350 1-351 1-352 1-353 1-3541-355 1-356 1-357 1-358 1-359 1-360 1-361 1-362 1-363 1-364 1-365 1-3661-367 1-368 1-369 1-370 1-371 1-372 1-373 1-374 1-375 1-376 1-377 1-3781-379 1-380 1-381 1-382 1-383 1-384 1-385 1-386 1-387 1-388 1-389 1-3901-391 1-392 1-393 1-394 1-395 1-396 1-397 1-398 1-399 1-400 1-401 1-4021-403 1-404 1-405 1-406 1-407 1-408 1-409 1-410 1-411 1-412 1-413 1-4141-415

Methods of Manufacture

In one embodiment, expression, isolation, purification and refolding(e.g., of an IL-10 delivery construct) can be performed according to theprocess outlined in FIG. 2A. In another embodiment, expression,isolation, purification and refolding (e.g., of an IL-10 deliveryconstruct) can be performed according to the process outline in FIG. 2B.

In step 201 in FIG. 2A or step 301 in FIG. 2B cells are engineered andcultured to recombinantly express an IL-10 delivery construct, such asSEQ ID NO: 5, by transforming the cells with a plasmid encoding theIL-10 delivery construct. In some embodiments, the plasmid includes anucleic acid corresponding to the sequence in SEQ ID NO. 10 (or asequence having at least 90%, at least 92%, at least 95%, at least 98%,or at least 99% sequence identity thereto), which is a codon-improvedsequence for expression in bacteria. The plasmid can further comprise amarker for antibiotic resistance. The antibiotic to which the plasmidcan confer resistance can be kanamycin, ampicillin, tetracycline, orchloramphenicol. In some instances, the cells are bacterial cells. Thebacterium can be Escherichia coli. Transformed cells can further beexpanded. The expansion of the transformed cells can be clonalexpansion. The expanded cells can be transferred into a productionbioreactor for fermentation. The fermentation can occur in a 1500 Lbioreactor. In some embodiments, the fermentation occurs in the presenceof the antibiotic to which the plasmid confers resistance. Productionfermentation can comprise a cell growth phase followed by an expressionphase. The expression phase can comprise the use of isopropylβ-D-1-thiogalactopyranoside (IPTG) as an inducer. The IL-10 deliveryconstruct can be expressed intracellularly as insoluble inclusionbodies. At the end of production, the cells can be harvested bycentrifugation. This centrifugation can produce a first pelletcomprising the cells. The first pellet can be resuspended in a firstbuffer. The first buffer can comprise from 40 mM to 60 mM of Tris,preferably 50 mM. The first buffer can range from a pH of 7.5 to 8.5,preferably a pH of 8.0. The first buffer can further comprise from 15 mMto 25 mM of EDTA, preferably 20 mM EDTA. The weight ratio of cells inthe first pellet to first buffer can be from 1:4 to 1:6, preferably 1:5.The first pellet can be mixed in the first buffer for from 50 to 70minutes, preferably 60 minutes, until a homogenous mixture is obtained.

In step 202 in FIG. 2A or step 302 in FIG. 2B, the cultured cells aredisrupted e.g., by lysing to release the inclusion bodies. The lysingcan comprise high-pressure homogenization. The high-pressurehomogenization can occur in a microfluidizer. The high-pressurehomogenization can occur from 16,000 to 20,000 psi, or about 18,000 psi.Two rounds of lysis can occur in order to ensure that substantially allcells have been lysed. The lysed cells can be centrifuged from 6000 to10,000 rpm, or about 8000 rpm. Centrifugation can occur for 30 to 50minutes, or about 40 minutes and can produce a second pellet.

The supernatant can be removed, and the second pellet can be resuspendedin a second buffer. The second buffer can comprise from 40 mM to 60 mMof Tris, preferably 50 mM. The second buffer can range from a pH of 7.5to 8.5, preferably a pH of 8.0. The second buffer can further comprisefrom 15 mM to 25 mM of EDTA, preferably 20 mM EDTA. The second buffercan further comprise from 2% to 3% of Trion X-100, preferably 2.5%. Thesecond buffer can further comprise from 450 mM to 550 mM of NaCl,preferably 500 mM. The weight ratio of the second pellet to secondbuffer can be from 1:4 to 1:6, preferably 1:5. The resuspension of thesecond pellet in the second buffer can be centrifuged from 6000 to10,000 rpm, or about 8000 rpm. Centrifugation can occur for 15 to 25minutes, or about 20 minutes and can produce a third pellet.

The supernatant can be removed, and the third pellet can be resuspendedin a third buffer. The third buffer can comprise from 40 mM to 60 mM ofTris, preferably 50 mM. The third buffer can range from a pH of 7.5 to8.5, preferably a pH of 8.0. The third buffer can further comprise from15 mM to 25 mM of EDTA, preferably 20 mM EDTA. The weight ratio of thethird pellet to third buffer can be from 1:4 to 1:6, preferably 1:5. Theresuspension of the third pellet in the third buffer can be centrifugedfrom 6000 to 10,000 rpm, or about 8000 rpm. Centrifugation can occur for15 to 25 minutes, or about 20 minutes and can produce a fourth pellet.

The supernatant can be removed, and the fourth pellet can be resuspendedin a fourth buffer. The fourth buffer can comprise from 40 mM to 60 mMof Tris, preferably 50 mM. The fourth buffer can range from a pH of 7.5to 8.5, preferably a pH of 8.0. The weight ratio of the fourth pellet tofourth buffer can be from 1:4 to 1:6, preferably 1:5. The resuspensionof the fourth pellet in the fourth buffer can be centrifuged from 6000to 10,000 rpm, or about 8000 rpm. Centrifugation can occur for 35 to 55minutes, or about 45 minutes and can produce a fifth pellet. The fifthpellet can comprise the inclusion bodies comprising the IL-10 deliverycomplex. The fifth pellet comprising the IL-10 delivery constructs canbe frozen prior to further use. The constructs can be frozen from −15°C. to −25° C., preferably −20° C.

In step 203 in FIG. 2A or step 303 in FIG. 2B, the inclusion bodies withthe IL-10 delivery construct are solubilized using a solubilizationsolution. The solubilization solution can comprise a chaotropic agent.The solubilization solution can comprise the chaotropic agent in aconcentration from 5 M to 8 M, from 6 M to 7 M, about 6.6 M, or about 6M. The chaotropic agent can comprise guanidine hydrochloride, urea, or acombination thereof. The chaotropic agent can comprise a hydrochloridesalt of guanidine. The solubilization solution can further compriseTris. The solubilization solution can comprise Tris in a concentrationfrom 40 mM to 60 mM, or about 50 mM. The solubilization solution can beat a pH from 7 to 9 or at about 8. The solubilization solution can beadded to the pellet comprising the 10 delivery constructs obtainedfollowing the lysing of the cell. A ratio of the pellet comprising theIL-10 delivery constructs to the solubilization solution can be from 1:8to 1:12 or at about 1:10 (w/w). The solubilization can be allowed to mixfor at least or about 60 mins.

In some embodiments, as shown in step 204 in FIG. 2A, the IL-10 deliveryconstruct is modified by a sulfitolysis agent or a reducing agent. Suchmodification may occur concurrent with or subsequent to solubilizationof the inclusions bodies as depicted in step 203. In some instances, asulfitolysis agent or a reducing agent is added to the solubilizationsolution prior to contacting the inclusion bodies with thesolubilization solution. In such instances, the solubilization andsulfitolysis/reduction steps may occur at the same time. In otherembodiments, the inclusion bodies are first solubilized in asolubilization solution, and the sulfitolysis agent or reducing agent issubsequently added. Stated differently, the sulfitolysis or reducingagent may be added after the IL-10 delivery constructs has beensubstantially solubilized. In some embodiments, the sulfitolysis agentcomprises sodium sulfite. For instance, in some embodiments, the cancomprise adding sodium sulfite to the solubilization solution. In someembodiments, from 30 mM to 50 mM, from 35 mM to 45 mM, from 38 mM to 42mM, or about 40 mM of sodium sulfite is added to the solubilizationsolution. In some embodiments, the method comprises incubating thesolubilization solution comprising the sodium sulfite for from 25 to 35minutes or more preferably for about 30 minutes. The incubating thesolubilization solution comprising the sodium sulfite can occur at roomtemperature. Potassium tetrathionate can then be added to thesolubilization solution. The potassium tetrathionate can be added to thesolubilization solution after addition of the sodium sulfite. In someembodiments, from 23 mM to 43 mM, from 28 mM to 38 mM, from 31 mM to 35mM, or about 33 mM of potassium tetrathionate is added to thesolubilization solution. Potassium tetrathionate can be mixed with thesolubilization solution for from 55 to 65 minutes or about 60 minutes.This mixing and incubation can occur at room temperature. Higher yieldsof an IL-10 delivery construct in dimer form may be obtained when asulfitolysis agent is used for disruption of disulfide bonds relative towhen DTT is used for disruption of disulfide bonds. For example, use ofthe sulfitolysis agent may result, upon refolding, in a yield of theIL-10 delivery construct in a dimer form that is at least 2-fold higherthan the yield obtained, after refolding, when DTT is used forreduction/disruption. For example, when IL-10 delivery constructs areprocessed using DTT for reduction, less than 5% of the resulting yieldof IL-10 delivery constructs may be in dimer form, whereas IL-10delivery constructs processed using a sulfitolysis agent may result ingreater than 10% of the resulting yield of IL-10 delivery constructs indimer form.

In some embodiments, step 204 in FIG. 2A is optional (or explicitlyabsent). Stated differently, in some embodiments, the solubilized IL-10,IL-10 delivery constructs, or solubilized inclusion bodies containingIL-10 or IL-10 delivery constructs are processed (e.g., clarified,concentrated, and/or delivered to a refolding solution) withouttreatment or contact with a reducing agent or a sulfitolysis agent (FIG.2B). In other words, in some embodiments, the inclusion bodies (IBs) aresolubilized with a chaotrophic agent and subsequently diluted into arefolding solution (e.g., a redox cocktail) without subjecting theinclusion bodies to a reducing agent or a sulfitolysis agent.

The method can comprise clarifying the solubilized and/or reduced IL-10delivery constructs to produce a clarified IL-10 delivery constructs(step 205 in FIG. 2A or step 304 in FIG. 2B). Clarification can compriseremoval of residual insoluble material following the solubilization andsulfitolysis and can occur prior to subsequent downstream purificationsteps. The clarifying can comprise depth filtration. The clarifying cancomprise a primary clarification. The primary clarification can comprisefiltering solubilized and/or the reduced IL-10 delivery constructsthrough a filter with a 0.5 μm to 10 μm nominal rating. The clarifyingcan comprise a secondary clarification. The secondary clarification canoccur after the primary clarification. The secondary clarification cancomprise filtering the solubilized and/or reduced IL-10 deliveryconstructs through a filter, such as a filter with a 0.2 μm to 2 μmnominal rating. The method can further comprise performing a sterilefiltration of the solubilized and/or reduced IL-10 delivery constructs.The sterile filtration can comprise filtration through a filter with apore size from 0.1 μm to 0.3 μm. The filter can be a capsule filter. Theperforming the sterile filtration can occur after the clarifying.

The method can comprise performing a tangential flow filtration stepbetween the clarification and refolding steps. The tangential flowfiltration step between the clarification and refolding steps cancomprise the first tangential flow filtration (TFF-1) of step 206 inFIG. 2A. The method can comprise performing a tangential flow filtrationstep between the clarification and refolding steps of the solubilizedand/or reduced IL-10 delivery constructs. In some embodiments, when asulfitolysis agent is not used, the method does not comprise atangential flow filtration step between the clarification and refoldingsteps (FIG. 2B). Stated differently, in some embodiments, step 206 isexplicitly absent when step 205 is also absent (FIG. 2A).

In some cases, purification without use of a sulfitolysis agent or areducing agent produces a higher IL-10 delivery construct dimerpercentage compared to solubilization using sulfitolysis or a reducingagent. In some cases, purification without use of sulfitolysis or areducing agent produces a higher IL-10 delivery construct yield comparedto solubilization by sulfitolysis or with a reducing agent. In somecases, purification without use of a sulfitolysis agent or a reducingagent produces fewer IL-10 delivery construct HMW aggregates compared tosolubilization using sulfitolysis or a reducing agent.

Furthermore, in some embodiments, purification without the use of asulfitolysis agent or a reducing agent does not require a tangentialflow filtration step between the clarification and refolding steps,which can shorten the purification process by about 1 or 2 days. Notperforming the tangential flow filtration step between the clarificationand refolding steps can prevent a loss of from 10% to 40%, from 10% to15%, from 15% to 30%, or from 30% to 35% of the purified IL-10 deliveryconstruct relative to a purification process including sulfitolysis anda tangential flow filtration step between the clarification andrefolding steps.

The first tangential flow filtration step between the clarification andrefolding steps can occur after the clarifying. The tangential flowfiltration step between the clarification and refolding steps can occurafter the sterile filtration of the solubilized and/or reduced IL-10delivery constructs. The tangential flow filtration step between theclarification and refolding steps can comprise ultrafiltration. Theultrafiltration can comprise concentration of the IL-10 deliveryconstructs to from 15 mg/mL to 25 mg/mL, from 18 mg/mL to 22 mg/mL, orabout 20 mg/mL. The ultrafiltration can occur at occur at atransmembrane pressure (TMP) from 10 to 20 psi, from 12 to 18 psi, orabout 15 psi. The tangential flow filtration step between theclarification and refolding steps can comprise diafiltration. Thediafiltration can occur after the ultrafiltration. The tangential flowfiltration step between the clarification and refolding steps cancomprise ultrafiltration and diafiltration (UF/DF). The diafiltrationcan comprise a first diavolume, a second diavolume, a third diavolume, afourth diavolume, and a fifth diavolume. The first diavolume, seconddiavolume, third diavolume, fourth diavolume, and fifth diavolume cancomprise a buffer. The buffer can comprise a chaotropic agent. Thebuffer can comprise from 3.5 M to 4.5 M of the chaotrophic agent,preferably 4 M. The chaotropic agent can be guanidine HCl. The buffercan comprise Tris. The buffer can comprise from 40 mM to 60 mM Tris,preferably 50 mM. The buffer can have a pH from 7 to 8.5. Thediafiltration can occur at occur at a transmembrane pressure (TMP) from10 to 20 psi, from 12 to 18 psi, or about 15 psi.

The method can comprise contacting the solubilized and/or reduced IL-10delivery constructs with a refolding solution to produce a refoldedIL-10 delivery constructs (step 207 in FIG. 2A or step 305 in FIG. 2B).The solubilized and/or reduced IL-10 delivery constructs can be in aretentate obtained following the tangential flow filtration step betweenthe clarification and refolding steps of the solubilized and/or reducedIL-10 delivery constructs. The refolding solution can comprise reducedglutathione and oxidized glutathione. The ratio (w/w) of reducedglutathione to oxidized glutathione can be from 0.8:1 to 1.2:1,preferably 1:1. The molar ratio of reduced glutathione to oxidizedglutathione can be from 0.8:2 to 1.1:2, preferably 1:2. In someembodiments, the refolding solution comprises from 0.75 mM to 1.5 mMreduced glutathione, preferably 1.0 mM. In some embodiments, therefolding solution comprises from 0.25 mM to 0.75 mM oxidizedglutathione, preferably 0.5 mM. In some embodiments, the refoldingsolution comprises arginine, sucrose, Tris, EDTA, or a combinationthereof. The refolding solution can comprise from 900 mM to 1.1 M ofarginine, preferably 1M. In some embodiments, the arginine isarginine-HCl. The refolding solution can comprise from 200 mM to 300 mMof sucrose, preferably 250 mM. The refolding solution can comprise from75 mM to 125 mM of Tris, preferably 100 mM. The Tris can have a pH ofabout 8.5. The refolding solution can comprise from 1.75 mM to 2.25 mMof EDTA, preferably 2 mM. In some embodiments, the refolding solutioncomprises polyethylene glycol (PEG). In some embodiments from 0.1% to0.3% (w/w) of the refolding solution is polyethylene glycol (PEG),preferably 0.2%. The PEG can be PEG 3350. The refolding solution cancomprise a pH from about 7.5 to about 8.5. The refolding solution cancomprise a pH of about 8.0. The refolding solution can comprise a pH ofabout 8.5. The retentate obtained following the tangential flowfiltration step between the clarification and refolding steps can bemixed with the refolding solution over the course of from 50 to 70minutes, preferably 60 minutes, to reach a target concentration of theIL-10 delivery constructs of from 0.8 mg/mL to 1.2 mg/mL, preferably 1mg/mL. Subsequent contacting with the refolding solution can occur from12 hours to 18 hours. The contacting with the refolding solution canoccur for at least 16 hours. The refolding solution can be at atemperature from 2° C. to 8° C., or at about 4° C., during thecontacting. The refolding solution can be pre-chilled to a temperaturefrom 2° C. to 8° C., or at about 4° C., prior to the contacting. Thecontacting can produce refolded IL-10 delivery constructs.

The method can comprise performing a first sterile filtering of therefolded IL-10 delivery constructs. The first sterile filtering cancomprise filtration through a filter with a pore size from 0.1 μm to 0.3μm, preferably 0.2 μm. The filter can be a capsule filter. The firststerile filtering of the refolded IL-10 delivery constructs can occurprior to a tangential flow filtration step between the refolding andanion exchange (AEX) chromatography steps.

In some embodiments, IL-10 delivery construct dimers may be stored inbuffer, for example at 25° C. for two days. Such a buffer may comprise asalt such as 1×PBS, 150 mM, or 200 mM NaCl buffered in 10 mM SodiumPhosphate at pH 7.0. IL-10 delivery construct dimers may be more stablewhen stored in a buffer comprising a salt such as 1×PBS, 150 mM, or 200mM NaCl buffered in 10 mM Sodium Phosphate at pH 7.0 than in a buffercomprising 10 mM Sodium Phosphate at pH 7.0 alone.

The method can comprise performing a tangential flow filtration stepbetween the refolding and anion exchange (AEX) chromatography steps. Thetangential flow filtration step between the refolding and anion exchange(AEX) chromatography steps can comprise the second tangential flowfiltration (TFF-2) of step 208 in FIG. 2A or the first tangential flowfiltration (TFF-1) of step 306 of FIG. 2B. The tangential flowfiltration step between the refolding and anion exchange (AEX)chromatography steps can occur after the first sterile filtering. Thetangential flow filtration step between the refolding and anion exchange(AEX) chromatography steps can comprise ultrafiltration. Theultrafiltration can occur at occur at a transmembrane pressure (TMP)from 10 to 20 psi, from 12 to 18 psi, or about 15 psi. The tangentialflow filtration step between the refolding and anion exchange (AEX)chromatography steps can comprise diafiltration. The diafiltration canoccur after the ultrafiltration. The tangential flow filtration stepbetween the refolding and anion exchange (AEX) chromatography steps cancomprise ultrafiltration and diafiltration (UF/DF). The diafiltrationcan comprise a first diavolume, a second diavolume, a third diavolume,and a fourth diavolume. The first diavolume and the second diavolume cancomprise a cold buffer (e.g., from 2-8 degrees C., or at about 4° C.).The third diavolume and the fourth diavolume can comprise a roomtemperature buffer. The cold buffer and the room temperature buffer cancomprise Tris and NaCl. The Tris can be in a concentration from 20 mM to30 mM, preferably 25 mM. The NaCl can be in a concentration from 75 mMto 125 mM, preferably 100 mM. The cold buffer and the room temperaturebuffer can be at a pH from 7 to 8, preferably 7.5. The retentateobtained following the tangential flow filtration step between therefolding and anion exchange (AEX) chromatography steps can be heldovernight at room temperature. The retentate obtained following thetangential flow filtration step between the refolding and anion exchange(AEX) chromatography steps can be held overnight from 2° C. to 8° C. orat about 4° C. The retentate obtained following the tangential flowfiltration step between the refolding and anion exchange (AEX)chromatography steps can comprise the refolded IL-10 delivery construct.

The method can comprise performing a second sterile filtering of therefolded IL-10 delivery construct. The second sterile filtering cancomprise filtering the retentate obtained following the tangential flowfiltration step between the refolding and anion exchange (AEX)chromatography steps through a filter with a pore size from 0.1 μm to0.3 μm. The filter can be a capsule filter. The second sterile filteringof the refolded IL-10 delivery construct can occur after the tangentialflow filtering step between the refolding and anion exchange (AEX)chromatography steps.

In some embodiments, the steps in the method, from refolding up to andincluding the tangential flow filtration step between the refolding andanion exchange (AEX) chromatography steps, are carried out at atemperature from 2° C. to 8° C. or from 3° C. to 5° C. In someembodiments, the steps in the method, from refolding up to and includingthe tangential flow filtration step between the refolding and anionexchange (AEX) chromatography steps, are carried out at a temperature ofabout 4° C. The method can comprise performing anion exchange (AEX)chromatography (step 209 in FIG. 2A or step 307 in FIG. 2B) on retentateobtained following the tangential flow filtration step between therefolding and anion exchange (AEX) chromatography steps. Performing AEXchromatography can comprise binding the IL-10 delivery construct dimersto an anion exchange column and subsequently eluting the IL-10 deliveryconstruct dimers from the anion exchange column. Performing AEXchromatography on the pool of IL-10 delivery constructs can therebycreate a first plurality of fractions of IL-10 delivery constructs. TheAEX chromatography can be Capto™ Q ImpRes.

The percentage of IL-10 delivery constructs in dimer form in eachfraction of the first plurality of fractions is determined using, forexample, size exclusion chromatography (SEC), such as size exclusionhigh performance liquid chromatography (SE-HPLC). The percentage ofIL-10 delivery constructs in a dimer form can be compared to a firstthreshold. The first threshold can be 70%, at least 75%, at least 80%,at least 85%, or at least 90%. Preferably, the first threshold can be75%. Any fraction containing a percentage of IL-10 delivery constructdimers greater than the threshold can be pooled into a first enrichedpool.

The method can comprise performing a ceramic hydroxyapatite (CHT)chromatography step on the first enriched pool (step 210 in FIG. 2A orstep 308 in FIG. 2B). Performing CHT chromatography on the firstenriched pool can thereby create a second plurality of fractions ofIL-10 delivery constructs in the dimer form. In some embodiments, theconcentration of the IL-10 delivery constructs in the second pluralityof fractions is from about 15 mg/mL to about 25 mg/mL or about 20 mg/mL.In some embodiments, the method does not comprise cation exchangechromatography. In some embodiments, the method does not comprise gelfiltration chromatography.

The percentage of IL-10 delivery constructs in dimer form in eachfraction of the second plurality of fractions is determined using, forexample, size exclusion chromatography (SEC), such as size exclusionhigh performance liquid chromatography (SE-HPLC). The percentage ofIL-10 delivery constructs in dimer form can be compared to a secondthreshold. The second threshold can be at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95%. Preferably, the secondthreshold can be 80%. Any fraction containing a percentage of IL-10delivery construct dimers greater than the threshold can be pooled intoa second enriched pool. The percentage of IL-10 delivery constructs inthe second enriched pool can be greater than the percentage of IL-10delivery constructs in the first enriched pool. The second enriched poolcan comprise greater than 80%, greater than 85%, or greater than 90% ofthe IL-10 delivery constructs in a dimer form.

The method can comprise a first sterile filtering of the second enrichedpool. The first sterile filtering of the second enriched pool cancomprise filtration through a filter, such as a filter with a pore sizefrom 0.1 μm to 0.3 μm. The filter can be a capsule filter.

The method can comprise performing a tangential flow filtration stepafter the ceramic hydroxyapatite chromatography step. The tangentialflow filtration step after the ceramic hydroxyapatite chromatographystep can comprise the third tangential flow filtration (TFF-3) of step211 in FIG. 2A or the second tangential flow filtration (TFF-2) of step309 of FIG. 2B. The tangential flow filtration step after the ceramichydroxyapatite chromatography step can comprise ultrafiltration. Theultrafiltration can occur at occur at a transmembrane pressure (TMP)from 10 to 20 psi, from 12 to 18 psi, or about 15 psi. The tangentialflow filtration step after the ceramic hydroxyapatite chromatographystep can comprise diafiltration. The diafiltration can occur after theultrafiltration. The tangential flow filtration step after the ceramichydroxyapatite chromatography step can comprise ultrafiltration anddiafiltration (UF/DF). The diafiltration can comprise a first diavolume,a second diavolume, a third diavolume, a fourth diavolume, and a fifthdiavolume. The first diavolume, second diavolume, third diavolume,fourth diavolume, and fifth diavolume can comprise a buffer. The buffercan be a lyophilization buffer. The buffer can comprise a salt, abulking agent, and an osmolyte. The buffer can comprise from 8 mM to 12mM salt, preferably 10 mM. The buffer can comprise from 1% to 3% bulkingagent, preferably 2%. The buffer can comprise from 0.5% to 1.5%osmolyte, preferably 1%. The salt can be potassium phosphate. Thebulking agent can be glycine. The osmolyte can be sucrose. The methodcan comprise a second sterile filtering. The second sterile filteringcan be performed after the diafiltration. The method can comprise addinga surfactant to the buffer. The surfactant can be added after the secondsterile filtering. Following the addition of the surfactant to thebuffer, the buffer can comprise from 0.2% to 0.4% of the surfactant,preferably 0.3%. The surfactant can be a poloxamer. The poloxamer can bepoloxamer 188. In some embodiments, the mixture of the buffer with therefolded IL-10 delivery constructs can be the liquid compositionpreviously described herein. The tangential flow filtration step afterthe ceramic hydroxyapatite chromatography step can occur after the firststerile filtering of the second enriched pool. The method can compriseperforming a second sterile filtering of the second enriched pool. Thesecond sterile filtering can comprise filtration through a filter, suchas a filter with a pore size from 0.1 μm to 0.3 μm, preferably 0.2 μm.The filter can be a capsule filter. The second sterile filtering canoccur after the tangential flow filtration step after the ceramichydroxyapatite chromatography step. The retentate obtained following thethird tangential flow filtration can be frozen from −70° C. to −90° C.,preferably −80° C. The retentate obtained following the tangential flowfiltration step after the ceramic hydroxyapatite chromatography step canbe the liquid composition described herein. The retentate obtainedfollowing the third tangential flow filtration can comprise greater than80%, greater than 85%, or greater than 90% of the IL-10 deliveryconstructs in a dimer form.

In some cases, the method can comprise performing cation exchangechromatography, for example with a Sulfate 650F column. The cationexchange chromatography step may be performed after an anion exchangechromatography step and a ceramic hydroxyapatite (CHT) purificationstep, before an anion exchange chromatography step and a ceramichydroxyapatite (CHT) purification step, or between an anion exchangechromatography step and a ceramic hydroxyapatite (CHT) purificationstep. As shown in Example 39 and Table 61, performing a cation exchangechromatography step, followed by an anion exchange chromatography stepand a ceramic hydroxyapatite (CHT) purification step resulted inrecovery of 20% of the IL-10 delivery construct dimers with 96% purity.

Oral Formulations

The solutions herein comprising high levels of a dimer form of IL-10(whether alone or as part of an IL-10 delivery construct) can be furtherprocessed for oral administration.

First, such solutions can be dried by a process that does not involveconcentration of the IL-10 delivery construct in a solution, examples ofsuch a process include lyophilization (freeze-drying, (FD)) or spraydrying (SD), to produce a dry or solid form of the IL-10/IL-10 deliveryconstruct composition. Freeze-drying can be conducted using a VirtisAdvantage manifold lyophilizer, with Intellitronics software. Glassvials containing a frozen therapeutic protein formulation can bepartially stoppered with a neoprene lyo-stopper, and then placed intojars connected to the lyo manifold and under vacuum (e.g., 1-100milli-Torr, or less) for about 12-48 hours. The lyophilized compositioncan be used to produce a capsule or a tablet formulation. In someembodiments, greater than 80%, greater than 85%, or greater than 90% ofthe IL-10 in the lyophilized composition is in a dimer form.

A formulation comprising IL-10 can be delivered to the small intestinesor colon in a formulation described herein. The formulation can bedelivered orally or rectally. In some embodiments, such formulations mayfacilitate crossing of the construct across the intestinal epithelialcell barrier (e.g., via transcytosis), which can otherwise preventachievement of the full therapeutic potential of the IL-10. Furthermore,targeted delivery of IL-10 directly to gastrointestinal tissue via theoral route may bypass the side effects experienced with systemicadministration and can translate into higher mucosal concentrations andclinically meaningful reductions in inflammation and disease.

Coated Oral Formulations for Targeted Release in the GI Tract

Contemplated herein are oral formulations comprising a therapeuticpayload and one or more excipients providing an improved release profilethat allows for a selective delivery of any payload to a certain regionwithin the gastrointestinal (GI) tract of a subject. Preferably, theoral formulations are configured for site site-specific release of thetherapeutic payload in the terminal ileum, proximal colon, or distalcolon. EXAMPLE 13 describes coated oral formulations configured forsite-specific release in the GI tract.

Payloads contemplated herein can be of any nature, includingtherapeutic, diagnostic, and imaging. A payload can be part of adelivery construct. A delivery construct can include a carrier coupledto a heterologous payload. The payload can be directly or indirectly,covalently or non-covalently, coupled to the carrier. When covalentlyattached, a payload can be directly attached to a carrier or via aspacer. While in one embodiment the payload is a therapeutic proteinsuch as IL-10 or an IL-10 delivery construct (such as IL-10 deliveryconstructs described herein), the disclosure herein is not limited toany therapeutic protein, carrier, or payload.

The oral formulation for delivery of a payload, such as a therapeuticprotein, to the lower GI tract can comprise a capsule or tablet with acoating configured to dissolve at a pH found in the small intestines orcolon, which has a pH in the range of from about 5.5 to about 8.0. Insome embodiments, the coating is configured not to dissolve in thehighly acidic pH of the stomach, which can range from a pH of about 1.5to about 3.5.

An oral formulation herein can be configured to pass through the stomachwithout releasing the payload to an appreciable extent. Release of thepayload can occur after full or partial dissolution of at least onecoating on a capsule or tablet comprising the payload. Release of thepayload can occur after damage to a capsule or tablet, includingmicroscopic damage such that the capsule or tablet can appear intact. Insome embodiments, the oral formulation is configured to release lessthan about 5%, less than about 4%, less than about 3%, less than about2%, less than about 1%, or 0% of the payload in the stomach. In someembodiments, the oral formulation is configured to release the payloadin specific regions within the small intestine or the colon, such as theterminal ileum, proximal colon, and distal colon. The terminal ileum, orthe distal end of the small intestines, intersects with the colon, andinflammation at this location can often be associated with GI disorderssuch as Crohn's disease. Site-specific release of therapeutic payloadswith anti-inflammatory properties in the terminal ileum can therefore bedesirable as a way to treat such disorders. The oral formulation can beconfigured to release from about 20% to 100% of the therapeutic payloadupon exposure to a solution at a pH from about 6.5 to about 7.0 for from2 to 8 hours. The solution can be citrate/phosphate buffer at theappropriate pH. The solution can be a digestive fluid. The digestivefluid can be stomach acid, intestinal juice (succus entericus), or acombination thereof. The digestive fluid can comprise digestive enzymes.The digestive fluid can be found in the stomach, small intestine, colon,or a combination thereof.

In some embodiments, the oral formulation is configured to release from80% to 100% of the therapeutic payload upon exposure to a solution at apH from about 6.9 to about 7.1, preferably a pH of 7.0, for from 2 to 8hours The oral formulation can be configured to release from 75% to100%, from 75% to 85%, or from 85% to 95% of the therapeutic payloadupon exposure to a solution at a pH from about 6.9 to about 7.1,preferably a pH of 7.0, for 2 hours. The oral formulation can beconfigured to release at least 80%, 85%, 90%, or 95% of the therapeuticpayload upon exposure to a solution at a pH from about 6.9 to about 7.1,preferably a pH of 7.0, for 2 hours. In some cases, the exposure to thesolution may be conducted at 37° C.

In some embodiments, the oral formulation is configured to release from80% to 100% of the IL-10 upon exposure to a solution at a pH from about6.9 to about 7.1, preferably a pH of 7.0, for from 2 to 8 hours. Theoral formulation can be configured to release from 75% to 100%, from 75%to 85%, or from 85% to 95% of the IL-10 upon exposure to a solution at apH from about 6.9 to about 7.1, preferably a pH of 7.0, for 2 hours. Theoral formulation can be configured to release at least 80%, 85%, 90%, or95% of the IL-10 upon exposure to a solution at a pH from about 6.9 toabout 7.1, preferably a pH of 7.0, for 2 hours. In some cases, theexposure to the solution may be conducted at 37° C.

In some embodiments, the oral formulation is configured to release from50% to 100% of the therapeutic payload upon exposure to a solution at apH from about 6.4 to about 6.6, preferably a pH of 6.5, for about 2 to 8hours. The oral formulation can be configured to release from 50% to95%, from 60% to 70%, or from 75% to 90% of the therapeutic payload uponexposure to a solution at a pH from about 6.4 to about 6.6, preferably apH of 6.5, for 2 or 3 hours. The oral formulation can be configured torelease at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the therapeuticpayload upon exposure to a solution at a pH from about 6.4 to about 6.6,preferably a pH of 6.5, for 2 or 3 hours. In some cases, the exposure tothe solution may be conducted at 37° C.

In some embodiments, the oral formulation is configured to release from50% to 100% of the IL-10 upon exposure to a solution at a pH from about6.4 to about 6.6, preferably a pH of 6.5, for about 2 to 8 hours. Theoral formulation can be configured to release from 50% to 95%, from 60%to 70%, or from 75% to 90% of the IL-10 upon exposure to a solution at apH from about 6.4 to about 6.6, preferably a pH of 6.5, for 2 or 3hours. The oral formulation can be configured to release at least 60%,65%, 70%, 75%, 80%, 85%, or 90% of the IL-10 upon exposure to a solutionat a pH from about 6.4 to about 6.6, preferably a pH of 6.5, for 2 or 3hours. In some cases, the exposure to the solution may be conducted at37° C.

In some embodiments, the oral formulation is configured to release from20% to 100% of the therapeutic payload upon exposure to a solution at apH from about 5.9 to about 6.1, preferably a pH of 6.0, for about 2 to 8hours. The oral formulation can be configured to release from 20% to80%, or from 20% to 30%, of the therapeutic payload upon exposure to asolution at a pH from about 5.9 to about 6.1, preferably a pH of 6.0,for 2 or 3 hours. The oral formulation can be configured to release atleast 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of thetherapeutic payload upon exposure to a solution at a pH from about 5.9to about 6.1, preferably a pH of 6.0, for 2 or 3 hours. In some cases,the exposure to the solution may be conducted at 37° C.

In some embodiments, the oral formulation is configured to release from20% to 100% of the IL-10 upon exposure to a solution at a pH from about5.9 to about 6.1, preferably a pH of 6.0, for about 2 to 8 hours. Theoral formulation can be configured to release from 20% to 80%, or from20% to 30%, of the IL-10 upon exposure to a solution at a pH from about5.9 to about 6.1, preferably a pH of 6.0, for 2 or 3 hours. The oralformulation can be configured to release at least 20%, 25%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% of the IL-10 upon exposure to a solutionat a pH from about 5.9 to about 6.1, preferably a pH of 6.0, for 2 or 3hours. In some cases, the exposure to the solution may be conducted at37° C.

The oral formulation can be a solid. The oral formulation can comprise alyophilized composition or a spray dried composition. The lyophilizedcomposition or a spray dried composition can comprise the therapeuticprotein and the one or more excipients. The lyophilized composition or aspray dried composition can be a powder. The lyophilized composition ora spray dried composition can comprise microparticles. Themicroparticles can have a diameter of about 1 μm to about 500 μm, about5 μm to about 250 μm, about 5 μm to about 100 μm, about 5 μm to about 50μm, or about 5 μm to about 15 μm. The lyophilized composition or a spraydried composition can comprise granules. The solid oral formulation canbe a capsule. The capsule can encapsulate the lyophilized composition.The solid oral formulation can be a tablet. The oral formulation can bein a unit dose form.

The oral formulation can comprise from about 1 mg to about 5 mg, fromabout 1 mg to about 10 mg, from about 1 mg to about 20 mg, from about 20mg to about 50 mg, from about 20 mg to about 100 mg, or from about 50 mgto about 100 mg of the therapeutic protein. The oral formulation cancomprise about 1 mg, 5 mg, or 20 mg of therapeutic protein. In someembodiments, from about 32% to about 42% (w/w) of the lyophilizedcomposition is the therapeutic protein.

The one or more excipients can comprise, consist essentially of, orconsist of a surfactant, an osmolyte, a bulking agent, a salt, or acombination thereof. The one or more excipients can comprise, consistessentially of, or consist of potassium phosphate, glycine, sucrose, andpoloxamer 188. The one or more excipients can further comprise acompacting excipient.

In some embodiments, the one or more excipients can be an osmolyte.Osmolytes can be used in pharmaceutical formulations comprising proteinsto improve stability of the proteins and decrease protein aggregation.The osmolyte can be an amino acid (e.g. proline or glycine), amethyl-amine (e.g., betaine or trimethylamine-N-oxide), or a polyol orsugar (e.g. sorbitol or sucrose). The osmolyte can be sucrose,trehalose, glycine, mannitol, histidine, dextose/dextran, arginine,maltose, sorbitol, taurine, glycine betaine, sarcosine, raffinose,glycerol, proline, fructan, L-glutamate, lactose, or a combinationthereof. The osmolyte can be sucrose. The oral formulation can comprisea weight ratio of the osmolyte to therapeutic protein from about 0.3:1to about 0.7:1, from about 0.4:1 to about 0.6:1, from about 0.45:1 toabout 0.55:1, from about 0.49:1 to about 0.51:1, or more preferablyabout 0.5:1. In some embodiments, from about 15% to about 21% (w/w) ofthe lyophilized composition is the osmolyte.

In some embodiments, the one or more excipients can include asurfactant. Surfactants can be used in solid oral formulationscomprising proteins, such as a capsule or tablet, to enhancedisintegration of the solid oral formulation and increase solubility ofthe proteins. The surfactant can be polysorbate 80, polysorbate 20,poloxamer 188, or a combination thereof. The oral formulation cancomprise a weight ratio of the surfactant to therapeutic protein fromabout 0.1:1 to about 0.19:1, from about 0.12:1 to about 0.18:1, fromabout 0.14:1 to about 0.16:1, or more preferably about 0.15:1. In someembodiments, from about 4.5% to about 6.5% (w/w) of the lyophilizedcomposition is the surfactant. The surfactant can be a non-ioniccopolymer. The non-ionic copolymer can comprise a centralpolyoxypropylene chain flanked by two polyoxyethylene chains. Thenon-ionic copolymer can be a poloxamer. Use of a poloxamer as anexcipient in the compositions described herein can promote or maintaindimerization of the IL-10 or IL-10 delivery construct relative to theuse of other surfactants, such as a polysorbate.

The poloxamer can comprise a molecular mass of polyoxypropylene from1600 g/mol to 2000 g/mol. The poloxamer can comprise from 70% to 90%polyoxyethylene. The poloxamer can be poloxamer 188. In someembodiments, the surfactant is not a polysorbate, such as polysorbate 80(e.g. Tween 80) or polysorbate 20 (e.g. Tween 20). An IL-10 deliveryconstruct composition comprising a poloxamer as an excipient can have agreater amount of IL-10 in a dimer form relative to an IL-10 deliveryconstruct composition comprising a polysorbate as an excipient. An IL-10delivery construct composition comprising a poloxamer as an excipientcan have a decreased amount of IL-10 in an aggregate or monomer formrelative to an IL-10 delivery construct composition comprising apolysorbate as an excipient.

The one or more excipients can include a salt. The salt can be potassiumphosphate, sodium chloride, potassium chloride, magnesium chloride,sodium sulfate, or a combination thereof. The salt can be potassiumphosphate. The oral formulation can have a weight ratio of the salt totherapeutic protein from about 0.03:1 to about 0.1:1, from about 0.05:1to about 0.09:1, from about 0.06:1 to about 0.08:1, or more preferablyabout 0.07:1. In some embodiments, from about 2% to about 3% (w/w) ofthe lyophilized composition is the salt.

The one or more excipients can include sodium hydroxide. The oralformulation can have a weight ratio of the sodium hydroxide totherapeutic protein from about 0.03:1 to about 0.1:1, from about 0.05:1to about 0.09:1, from about 0.06:1 to about 0.08:1, or more preferablyabout 0.07:1. In some embodiments, from about 2% to about 3% (w/w) ofthe lyophilized composition is sodium hydroxide.

In some embodiments, the one or more excipients can include a bulkingagent. Bulking agents can be used to increase the size of an oralformulation for ease of manufacturing. The bulking agent can be starch,lactose, dextrin, glucose, sucrose, sorbitol, raffinose, trehalose,glycine, mannitol, or a combination thereof. The bulking agent can beglycine. The oral formulation can comprise a weight ratio of the bulkingagent to therapeutic protein from about 0.7:1 to about 1.3:1, from about0.8:1 to about 1.2:1, from about 0.9:1 to about 1.1:1, or morepreferably about 1:1. In some embodiments, from about 32% to about 42%(w/w) of the lyophilized composition is the bulking agent. In someembodiments, the buffering agent is an osmolyte.

The lyophilized composition can be stored from about 2° C. to about 8°C. The lyophilized composition can be stored from about −15° C. to about−25° C. The lyophilized composition can be stable from about 2° C. toabout 8° C. with ambient relative humidity for at least 12 months. Insome embodiments, when the therapeutic protein is in a dimer form, thelyophilized composition is stable if there is no more than a 1%, 2%, 3%,4%, 5%, 10%, 20%, or 25% decrease in the amount of therapeutic proteindimers after one year of storage at from about 2° C. to about 8° C. Insome embodiments, when the therapeutic protein is in a dimer form, thelyophilized composition is stable if there is no more than a 1%, 2%, 3%,4%, 5%, 10%, 20%, or 25% decrease in the amount of therapeutic proteindimers after one year of storage at from about −15° C. to about −25° C.In some embodiments, the lyophilized composition is compacted into atablet or filled in a capsule to produce the oral formulation describedherein.

Further described herein, in certain embodiments, are liquidcompositions comprising the therapeutic protein and the one or moreexcipients which can be lyophilized to produce the lyophilizedcomposition described herein. The therapeutic protein can be an IL-10delivery construct as described herein. The liquid composition cancomprise a lyophilization buffer and the IL-10 delivery construct. Theliquid composition can comprise from 15 mg/mL to 25 mg/mL of the IL-10delivery construct, preferably about 20 mg/mL. The liquid compositioncan comprise from 1.51 mg/mL to 1.91 mg/mL of potassium phosphate,preferably about 1.71 mg/mL. The liquid composition can comprise from 15mg/mL to 25 mg/mL of glycine, preferably about 20 mg/mL. The liquidcomposition can comprise from 8 mg/mL to 12 mg/mL of sucrose, preferablyabout 10 mg/mL. The liquid composition can comprise from 2.5 mg/mL to3.5 mg/mL of Poloxamer 188, preferably about 3 mg/mL. The liquidcomposition can have a pH from about 5.0 to about pH 8.0, from about 6.0to about 7.5, or from about 6.5 to about 7.5. The liquid composition canhave a pH of from about 7.4 to about 7.6.

The liquid composition can be stable from about −80° C. to about −60° C.with ambient relative humidity for at least 12 months. In someembodiments, there is no more than a 2% decrease in percentage of thetherapeutic protein in the dimer form of the liquid composition after 7days at 4° C. The liquid composition can be frozen to produce a frozentherapeutic protein composition. The liquid composition can be frozen ata temperature from about −85° C. to about −15° C. In some embodiments,the frozen therapeutic protein composition is thawed prior tolyophilization.

In some embodiments, lyophilized composition is encapsulated in acapsule. The capsule can be a size 000, 00, 0, 1, 2, 3, 4, or 5 capsule.The capsule can be a two-piece capsule. The capsule can be ahydroxypropyl methylcellulose (HPMC) capsule, also referred to as aHypromellose capsule.

Alternatively, lyophilized composition can be compressed under acompression force to produce a tablet. The compression force can rangefrom 1500 pound-force (lbf) to 4000 lbf or from 2000 lbf to 3500 lbf.The compression force can be 1500 lbf, 2000 lbf, 2500 lbf, 3000 lbf,3500 lbf, or 4000 lbf. The tablet can have a weight from 150 mg to 1000mg, from 150 mg to 500 mg, from 200 mg to 400 mg, from 150 mg to 250 mg,from 175 mg to 225 mg, or from 190 mg to 210 mg. The tablet can comprisea diameter from 0.2″ to 0.4″, from 0.25″ to 0.35″, or from 0.3″ to0.25″. The tablet can comprise from 1 mg to 5 mg, from 1 mg to 10 mg,from 1 mg to 20 mg, from 20 mg to 50 mg, from 20 mg to 100 mg, or from50 mg to 100 mg of the IL-10 delivery construct. The tablet can compriseabout 1 mg, about 5 mg, about 10 mg, about 20 mg, or about 30 mg of theIL-10 delivery construct. The tablet can comprise from 4.5 mg to 5.5 mg,from 9.5 mg to 10.5 mg, from 19 mg to 21 mg, or from 29 mg to 31 mg ofan IL-10 or IL-10 delivery construct. The tablet can comprise from 3 mgto 9 mg, from 4 mg to 8 mg, or from 5 mg to 7 mg strength of an IL-10 orIL-10 delivery construct. The tablet can be round, oblong, oval,circular, or any other suitable shape.

When the oral formulation is a tablet, the one or more excipients caninclude a compacting excipient. The one or more excipients can comprise1, 2, 3, 4, or more than 4 compacting excipients. The compactingexcipient can be a disintegrant, a binding agent, a lubricant, or acombination thereof. The oral formulation can comprise a weight ratio ofthe lyophilized composition previously described to the compactingexcipient of from about 0.8:3 to about 1.2:3, from about 0.9:3 to about1.1:3, from about 0.95:3 to about 1.05:3, or more preferably about 1:3.The IL-10 delivery construct can comprise from about 5% to about 15%(w/w) of the tablet. In some embodiments, the compacting excipient isnot part of the liquid composition. In some embodiments, the lyophilizedcomposition does not include the compacting excipient.

The compacting excipients can comprise a disintegrant. A disintegrantcan facilitate the dispersion or break up of an oral formulation. Thedisintegrant can comprise microcrystalline cellulose (MCC), silicifiedmicrocrystalline cellulose (SMCC), starch, sodium starch glycolate,veegum, bentonite, alginic acid, calcium alginate, croscarmellose sodium(crosslinked sodium carboxymethyl cellulose), crospovidone (crosslinkedpolyvinylpyrrolidone), or a combination thereof.

The compacting excipients can comprise a binding agent. A binding agentcan hold the components of an oral formulation together. The bindingagent can comprise a disaccharide, a polysaccharide, a protein, or apolymer. The disaccharide can be sucrose or lactose. The lactose can belactose monohydrate. The polysaccharide can be starch, cellulose, or aderivative thereof. The protein can be gelatin. The polymer can bepolyvinylpyrrolidone (PVP) or polyethylene glycol (PEG).

The compacting excipients can be a lubricant. A lubricant can reduceinterparticle friction and cohesion in an oral formulation. Thelubricant can comprise magnesium stearate, glyceryl behenate, glyceryldibehenate, sodium stearyl fumarate, stearic acid, talc, silica, calciumstearate, magnesium carbonate, hydrogenated oil, mineral oil,polyethylene glycol (PEG), glyceryl monostearate or a combinationthereof. The lubricant can be a non-ionic surfactant. The non-ionicsurfactant can be glyceryl behenate. The glyceryl behenate be glyceryldibehenate. In some embodiments, the lubricant is not an ionicsurfactant, such as magnesium stearate, sodium stearyl fumarate, andsodium laurisulfate. In some embodiments, the use of a non-ionicsurfactant, such as glyceryl behenate or glyceryl dibehenate, in thegeneration of a tablet results in an improved dissolution profile of thetablet relative to if an ionic surfactant, such as magnesium stearate,sodium stearyl fumarate, or sodium laurisulfate, is used in thegeneration of the tablet. Stated differently, the use of a non-ionicsurfactant, such as glyceryl behenate or glyceryl dibehenate, may resultin a formulation in which a higher concentration of the IL-10 deliveryconstruct remains in dimer form for a longer period of time relative toa corresponding formulation that uses an ionic surfactant, such asmagnesium stearate, sodium stearyl fumarate, or sodium laurisulfate.

In some embodiments, the one or more compacting excipients are combinedwith the lyophilized composition previously described prior tocompacting into a tablet or filling a capsule. In some embodiments, acomposition for compacting can comprise from about 6% to about 10% (w/w)of the lyophilized composition and from about 90% to about 94% (w/w) ofthe one or more compacting excipients. In some embodiments, acomposition for compacting can comprise from about 5% to about 15% (w/w)of the lyophilized composition and from about 85% to about 95% (w/w) ofthe one or more compacting excipients. The one or more contactingexcipients can comprise at least two disintegrants.

In some embodiments, granules are formed during the process of making anoral formulation. For instance, in some cases, there are two phases toproduction of tablets using a granulation process: an intragranular (IG)phase and an extragranular (EG) phase. In the intragranular phase, thelyophilized composition and a first subset of the one or more compactingexcipients can be blended and granulated with a binder to produce ablended composition which can then be compressed (e.g by rollercompaction) and milled into granules. In the extragranular phase, thedry granules can then be blended with a second subset of the one or morecompacting excipients and compressed into an oral formulation, such as atablet.

The granules produced in the intragranular phase can comprise a weightratio of the first subset of the one or more compacting excipients tothe lyophilized powder comprising the IL-10 delivery construct of fromabout 7:1 to about 11:1, from about 8:1 to about 10:1, or from about8.5:1 to about 9.5:1. In some embodiments, the first subset of the oneor more compacting excipients can comprise at least two disintegrants.In some embodiments, the first subset of the one or more compactingexcipients can comprise a weight ratio of a first disintegrant to asecond disintegrant of from about 21:1 to about 24:1, from about 22:1 toabout 23.5:1, or from about 22.3:1 to about 23:1. The first disintegrantcan be silicified microcrystalline cellulose (SMCC) or dicalciumphosphate/microcrystalline cellulose (DCP/MCC). The second disintegrantcan comprise crospovidone or croscarmellose sodium. In some embodiments,the first subset of the one or more compacting excipients comprises alubricant. The weight ratio of the at least two disintegrants to thelubricant in the first subset of the one or more excipients can be fromabout 61:1 to about 81:1, from about 66:1 to about 76:1, or from about71:1 to about 73:1. The lubricant can comprise glyceryl dibehenate. Thelubricant can comprise glyceryl behenate.

The extragranular phase can comprise a weight ratio of the granulesproduced in the intragranular phase to the second subset of the one ormore compacting excipients from about 2:1 to about 6:1, from about 3:1to about 5:1, or from about 3.5:1 to about 4.5:1. In some embodiments,the second subset of the one or more compacting excipients can compriseat least two disintegrants. In some embodiments, the second subset ofthe one or more compacting excipients can comprise a weight ratio of afirst disintegrant to a second disintegrant of from about 13.5:1 toabout 24:1, from about 16:1 to about 21.5:1, or from about 17.5:1 toabout 19:1. The first disintegrant can be SMCC or DCP/MCC. The seconddisintegrant can comprise crospovidone or croscarmellose sodium. In someembodiments, the second subset of the one or more compacting excipientscomprises a lubricant. The weight ratio of the at least twodisintegrants to the lubricant to in the second subset of the one ormore excipients can be from about 69:1 to about 89:1, from about 74:1 toabout 84:1, or from about 77:1 to about 81:1. The lubricant can compriseglyceryl behenate. The glyceryl behenate can comprise glyceryldibehenate. In some embodiments, the compacting excipients can comprise,consist essentially of, or consist of SMCC, crospovidone, and glycerylbehenate.

In some embodiments, the oral formulation can comprise a weight ratio ofthe one or more compacting excipients to the lyophilized composition offrom about 9:1 to about 14:1, from about 10:1 to about 13:1, or fromabout 11:1 to about 12:1. In some embodiments, the oral formulation cancomprise a weight ratio of a first disintegrant of the one or morecompacting excipients to the lyophilized composition of from about 8.8:1to about 12.8:1, from about 9.8:1 to about 11.8:1, or from about 10.4:1to about 11.2:1. In some embodiments, the oral formulation can comprisea weight ratio of the lyophilized composition to a second disintegrantof the one or more compacting excipients of from about 1.5:1 to about2.5:1, from about 1.75:1 to about 2.25:1, or from about 1.9:1 to about2.1:1.

In some embodiments, the oral formulation can comprise a weight ratio ofthe lyophilized composition to a lubricant of the one or more compactingexcipients of from about 5:1 to about 8.1:1, from about 5.5:1 to about8.1:1, or from about 6.2:1 to about 6.6:1.

The oral formulation can comprise a first coat comprising a firstcopolymer, a second copolymer, or a mixture of the first copolymer andthe second copolymer. The oral formulation can comprise a first coat ofHypromellose acetate succinate (HPMCAS or HPMC-AS). The first coat canhave a thickness substantially equivalent to from 20 mg to 200 mg, from20 mg to 40 mg, from 50 mg to 70 mg, from 115 mg to 135 mg, or from 175mg to 185 mg of the first coat on a size 1 capsule. The first coat canhave a thickness substantially equivalent to from 55 mg to 65 mg of thefirst coat on a size 1 capsule. The first coat can have a thicknesssubstantially equivalent to from 20 mg to 200 mg, from 20 mg to 40 mg,from 50 mg to 80 mg, from 115 mg to 135 mg, or from 175 mg to 185 mg ofthe first coat on a size 0 capsule. The first coat can have a thicknesssubstantially equivalent to from 70 mg to 80 mg of the first coat on asize 0 capsule. The first coat can have a mass from 20 mg to 200 mg,from 20 mg to 40 mg, from 50 mg to 80 mg, from 115 mg to 135 mg, or from175 mg to 185 mg.

The surface area for a size 1 capsule can be approximately 410 mm². Thesurface area for a size 0 capsule can be approximately 500 mm². A coatthickness of 0.15 mg/mm² can be equivalent to a 60 mg coat weight on asize 1 capsule or 75 mg coat weight on a size 0 capsule. In someembodiments, the capsule has a coat thickness of from 0.1 mg/mm2 to 0.2mg/mm², preferably 0.15 mg/mm². In some embodiments, the coat thicknesson a capsule can be from 4 mg/cm² to 20 mg/cm², from 4 mg/cm² to 6mg/cm², from 5 mg/cm² to 10 mg/cm², or from 5 mg/cm² to 20 mg/cm². Thecoat thickness can be a thickness of the first coat.

In some embodiments, the tablet has a coat thickness of from 0.1 mg/mm²to 0.2 mg/mm², from 0.1 mg/mm² to 0.5 mg/mm², or from 0.1 mg/mm² to 1.0mg/mm² preferably 0.15 mg/mm². In some embodiments, the coat thicknesson a tablet can be from 4 mg/cm² to 20 mg/cm², from 4 mg/cm² to 6mg/cm², from 5 mg/cm² to 10 mg/cm², or from 5 mg/cm² to 20 mg/cm². Thecoat thickness can be a thickness of the first coat.

The first copolymer can have an individual nominal dissolution ofpH>5.5. The nominal dissolution pH indicates the pH at which thecopolymer becomes soluble. The first copolymer can comprise methacrylicacid and ethyl acrylate. The first polymer can have a weight averagemolecular mass from 200,000 g/mol to 450,000 g/mol, or from 250,000g/mol to 400,000 g/mol, or from 280,000 g/mol to 370,000 g/mol, or from300,000 g/mol to 340,000 g/mol. The first polymer can comprise a ratioof free carboxyl groups to ester groups in the first copolymer is from0.8:1 and 1.2 to 1. The first copolymer can comprise a polymer offormula I, wherein x, y, and n are each greater than or equal to one.The first copolymer can comprise Eudragit® L 30 D-55.

The second copolymer can have a nominal dissolution at pH>7.0. Thesecond copolymer can comprise methacrylic acid, methyl methacrylate, andmethyl acrylate. The second polymer can have a weight average molecularmass from 160,000 g/mol to 400,000 g/mol or from 200,000 g/mol to360,000 g/mol, or from 240,000 g/mol to 320,000 g/mol, or from 260,000g/mol to 300,000 g/mol. The second polymer can comprise a ratio of freecarboxyl groups to ester groups in the second copolymer is from 0.8:1and 1.2 to 1. The second polymer can comprise a polymer of formula II,wherein x, y, z, and n are each greater than or equal to one. The secondcopolymer can comprise Eudragit® FS 30 D.

The second copolymer can be different from the first copolymer. Thenominal dissolution pH of a mixture of the first copolymer and thesecond copolymer can be different from the nominal dissolution pH of thefirst copolymer or second copolymer individually. For example, thenominal dissolution pH of a mixture of the first copolymer and thesecond copolymer can be a nominal dissolution between the nominaldissolutions of the first copolymer and the second copolymer. The firstcoat can comprise an equal amount of the second copolymer relative to anamount of the first copolymer. The first coat can comprise a greateramount of the second copolymer relative to an amount of the firstcopolymer. A weight ratio of the first copolymer to the second copolymercan be about or between any of 50:50, 45:55, 40:60, 35:65, 30:70, 25:75,20:80, 15:85, 10:90, 5:95, or 0:100. A weight ratio of Eudragit® L30D55:Eudragit® FS30D can be about 50:50, 45:55, 40:60, 35:65, 30:70, 25:75,20:80, 15:85, 10:90, 5:95, or 0:100.

A weight ratio of the first copolymer to the second copolymer in thefirst coat can be from 0:100 to 100:0. The weight ratio of the firstcopolymer to the second copolymer in the first coat can be from 45:55 to55:45, from 25:75 to 35:65, from 15:85 to 25:75, or from 15:85 to 0:100.

In some embodiments, a weight ratio of the first copolymer to the secondcopolymer in the first coat of from 45:55 to 55:45 with an equivalentcoating thickness from 40 mg to 70 mg on a size 1 capsule results inrelease of the therapeutic payload in the terminal ileum. In someembodiments, a weight ratio of the first copolymer to the secondcopolymer of 50:50 with a coating thickness of about 0.15 mg/mm² resultsin release of the therapeutic payload in the terminal ileum.

In some embodiments, a weight ratio of the first copolymer to the secondcopolymer in the first coat of from 15:85 to 25:75 with an equivalentcoating thickness from about 118 mg to 138 mg on a size 1 capsuleresults in release of the therapeutic payload in the distal colon.

In some embodiments, a weight ratio of the first copolymer to the secondcopolymer in the first coat of from 15:85 to 25:75 with an equivalentcoating thickness from 40 mg to 70 mg on a size 1 capsule results inrelease of the therapeutic payload in the proximal colon. In someembodiments, a weight ratio of the first copolymer to the secondcopolymer of 20:80 with a coating thickness of about 0.15 mg/mm² resultsin release of the therapeutic payload in the proximal colon.

The first coat can further comprise an anti-tacking agent, aplasticizer, a surfactant, or a combination thereof. The anti-tackingagent can be glycerol monostearate. The plasticizer can be triethylcitrate. The surfactant can be polysorbate 80.

In some embodiments, from 5% to 15% (w/w) of the first coat is a mixtureof the anti-tacking agent, the plasticizer, and the surfactant. In someembodiments, from 40% to 50% (w/w) of the first coat is the firstcopolymer. In some embodiments, from 40% to 50% (w/w) of the first coatis the second copolymer. In some embodiments, the weight ratio of thefirst copolymer and second copolymer to the mixture of the anti-tackingagent, the plasticizer, and the surfactant is from 8:1 to 10:1, from8.5:1 to 9.5:1, or from 8.8:1 to 9.2:1.

In some embodiments, from 5% to 15% (w/w) of the first coat is a mixtureof glycerol monostearate, triethyl citrate, and polysorbate 80. In someembodiments, from 40% to 50% (w/w) of the first coat is a firstcopolymer comprising methacrylic acid and ethyl acrylate. In someembodiments, from 40% to 50% (w/w) of the first coat is a secondcopolymer comprising methacrylic acid, methyl methacrylate, and methylacrylate.

As previously described, the oral formulation can comprise a first coatof Hypromellose acetate succinate (HPMCAS or HPMC-AS). The first coat ofHPMCAS can comprise a mixture of a first HPMCAS and a second HPMCAS. Thefirst HPMCAS can become soluble at a pH of greater than or equal to 6.8.The second HPMCAS can become soluble at a pH of greater than or equal to6.0. The first HPMCAS can comprise HPMCAS-HF. The second HPMCAS cancomprise HPMCAS-MF. The ratio of the first HPMCAS to the second HPMCAScan be from about 40:60 to about 60:40 or from about 45:55 to about55:45.

In some embodiments, the oral formulation can be an oral formulation asrepresented by FIG. 31A. The oral formulation 3200 can comprise acapsule or a tablet, wherein the capsule or tablet comprises an interiorregion 3201 comprising the therapeutic protein and the one or moreexcipients. The oral formulation 3200 can comprise a first coat 3203.The first coat 3203 can comprise a mixture of the first copolymer andthe second copolymer. The first coat 3203 can further comprise ananti-tacking agent, a plasticizer, and a surfactant. The oralformulation 3200 can further comprise a second coat 3202. The secondcoat 3202 can comprise hydroxypropyl methylcellulose (HPMC). The secondcoat can seal a seam of the capsule. In some embodiments, the capsule isa hydroxypropyl methylcellulose (HPMC) capsule. The oral formulation3200 can comprise a third coat 3204. The third coat 3204 can compriseHPMC. In some embodiments, the first coat 3203, the second coat 3202,the third coat, or the combination thereof are applied to the capsule ortablet by spray-coating. In some embodiments, a solution of HPMC with anHPMC concentration of from 6.5% to 8.5% (w/w) is spray coated onto thecapsule or tablet to apply the second coat 3202, third coat, or both thesecond coat 3202 and third coat. In some embodiments, the second coat3202 comprises from 9 mg to 13 mg, from 10 mg to 12 mg, or from 10.5 mgto 11.5 mg of HPMC on a size 0 capsule. Equivalent coat weights of thesecond coat 3202 can be applied to other capsule sizes. In someembodiments, the third coat 3204 comprises from 9 mg to 13 mg, from 10mg to 12 mg, or from 10.5 mg to 11.5 mg of HPMC on a size 0 capsule.Equivalent coat weights of the third coat 3204 can be applied to othercapsule sizes.

In some embodiments, the oral formulation can be an oral formulation asrepresented by FIG. 31B. The oral formulation 3205 can comprise acapsule or a tablet, wherein the capsule or tablet comprises an interiorregion 3201 comprising the therapeutic protein and the one or moreexcipients. The oral formulation 3200 can comprise a first coat 3203.The first coat 3203 can comprise a mixture of the first copolymer andthe second copolymer. The first coat 3203 can further comprise ananti-tacking agent, a plasticizer, and a surfactant. In someembodiments, the oral formulation 3205 does not comprise a second coator a third coat.

The oral formulation can have a shelf life of at least 3 months, atleast 6 months, at least 12 months, at least 18 months, or at least 24months. Shelf life may be assessed by storing a tablet for the indicatedtime period, removing the coating from the tablet, dissolving the innercore of the tablet and assessing the dimer percentage as describedherein.

The oral formulation can be configured to release from about 20% to 100%of the IL-10 upon exposure to a solution at a pH from about 6.5 to about7.0 for from 2 to 8 hours at 37° C. The solution can becitrate/phosphate buffer at the appropriate pH. The solution can be adigestive fluid. The digestive fluid can be stomach acid, intestinaljuice (succus entericus), or a combination thereof. The digestive fluidcan comprise digestive enzymes. The digestive fluid can be found in thestomach, small intestine, colon, or a combination thereof. The IL-10 canbe in the form of an IL-10 delivery construct.

In some embodiments, the oral formulation is configured to release from80% to 100% of the IL-10 upon exposure to a solution at a pH from about6.9 to about 7.1, preferably a pH of 7.0, for from 2 to 8 hours. Theoral formulation can be configured to release from 75% to 100%, from 75%to 85%, or from 85% to 95% of the IL-10 upon exposure to a solution at apH from about 6.9 to about 7.1, preferably a pH of 7.0, for 2 hours. Theoral formulation can be configured to release at least 80%, 85%, 90%, or95% of the IL-10 upon exposure to a solution at a pH from about 6.9 toabout 7.1, preferably a pH of 7.0, for 2 hours.

In some embodiments, the oral formulation is configured to release from50% to 100% of the IL-10 upon exposure to a solution at a pH from about6.4 to about 6.6, preferably a pH of 6.5, for about 2 to 8 hours. Theoral formulation can be configured to release from 50% to 95%, from 60%to 70%, or from 75% to 90% of the IL-10 upon exposure to a solution at apH from about 6.4 to about 6.6, preferably a pH of 6.5, for 2 or 3hours. The oral formulation can be configured to release at least 60%,65%, 70%, 75%, 80%, 85%, or 90% of the IL-10 upon exposure to a solutionat a pH from about 6.4 to about 6.6, preferably a pH of 6.5, for 2 or 3hours.

In some embodiments, the oral formulation is configured to release from20% to 100% of the IL-10 upon exposure to a solution at a pH from about5.9 to about 6.1, preferably a pH of 6.0, for about 2 to 8 hours. Theoral formulation can be configured to release from 20% to 80%, or from20% to 30%, of the IL-10 upon exposure to a solution at a pH from about5.9 to about 6.1, preferably a pH of 6.0, for 2 or 3 hours. The oralformulation can be configured to release at least 20%, 25%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% of the IL-10 upon exposure to a solutionat a pH from about 5.9 to about 6.1, preferably a pH of 6.0, for 2 or 3hours.

In some embodiments, the oral formulation is configured such that from20% to 30% of the IL-10 released upon exposure to the solution at a pHof from about 6.5 to 7.0 for 2 hours is in a dimer form. In someembodiments, the oral formulation is configured such that at least 15%,20%, 25%, or 30% of the IL-10 released upon exposure to the solution ata pH of from about 6.5 to 7.0 for 2 hours is in a dimer form. In someembodiments, the oral formulation is configured such that no more than50%, 60%, 70%, 80%, or 90% of the IL-10 released upon exposure to thesolution at a pH of from about 6.5 to 7.0 for 2 hours is in a dimerform. In some embodiments, following submersion of the oral formulationinto a solution at pH 7.0, a percentage of IL-10 in the dimer form is atleast 35%, 40%, 45%, or 50%.

The oral formulation comprising IL-10 delivery constructs can beformulated to have at least 15% of the IL-10 delivery constructs remainin the dimer form after a five-minute incubation with simulatedintestinal fluid (SIF)/pancreatin. The pancreatin assay comprisesincubating the oral formulation comprising the therapeutic protein withpancreatin (10 μg) in PBS (100 μL) at 37° C.

The oral formulation can comprise from 0.3 mg to 10 mg, from 0.3 mg to 5mg, from 0.3 mg to 3 mg, from 1 mg to 3 mg, from 1 mg to 5 mg, from 1 mgto 10 mg, from 1 mg to 20 mg, from 20 mg to 50 mg, from 20 mg to 100 mg,or from 50 mg to 100 mg of the IL-10 delivery constructs. The oralformulation can comprise 0.3 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7mg, 8 mg, 9 mg, 10 mg, or 20 mg of the IL-10 delivery constructs.

The oral formulation comprising an IL-10 delivery construct can have ashelf life of at least 3 months, at least 6 months, at least 12 months,at least 18 months, or at least 24 months. The oral formulationcomprising an IL-10 delivery construct can be stable at a specifiedtemperature (e.g., 2-8° C. or room temperature) for a specified periodof time (e.g., for at least 1 month, 3 months, at least 6 months, atleast 12 months, at least 18 months, or at least 24 months). Forinstance, the oral formulation comprising an IL-10 delivery constructcan have sufficient stability such that the percentage of IL-10 deliveryconstructs in a dimer form does not decrease by more than 1%, 2%, 3%,4%, or 5% when stored at a specified temperature (e.g., 2-8° C. or roomtemperature) for a specified period of time (e.g., for at least 1 month,3 months, at least 6 months, at least 12 months, at least 18 months, orat least 24 months). In some embodiments, the oral formulation issufficiently stable such that the level of dimers of the IL-10 deliveryconstruct in the oral formulation remains at greater than 80%, greaterthan 85%, or greater than 90% after the period of time has passed.

Described herein, in certain embodiments, are kits comprising at leastone of a unit dosage form of the oral formulation described herein. Theunit dosage forms can be presented in a pack, dispenser device, orbottle. The pack can comprise metal or plastic foil. An example of apack can include, but is not limited to, a blister pack. The bottle canbe a high-density polyethylene (HDPE) bottle. The bottle can furthercomprise an induction seal. The unit dosage forms can be packaged withinthe kit separately (e.g., in different units of a blister pack) ortogether (e.g., combined in a single container, such as a bottle). Thekit can further comprise instructions for using the unit dosage formsfor the treatment of a disorder causing inflammation. The kit cancomprise a notice associated with the container in a form prescribed bya governmental agency regulating the manufacture, use, or sale ofpharmaceuticals. The governmental agency can be the U.S. Food and DrugAdministration. The notice can be an approved product insert.

Treatment

Described herein, in certain embodiments, are methods of treating adisease or condition in an individual in need thereof. The individualcan be a mammal. The mammal can be a primate. The primate can be ahuman. The individual can be an individual diagnosed with, suspected ofhaving, or at risk of a disease or condition. The disease or conditioncan be a disease or condition resulting in inflammation of a tissue ofan individual, also referred to as an inflammatory disorder. The diseaseor condition causing inflammation can be disease or conditioncharacterized by a deficiency in IL-10 expression. Treating the diseaseor condition (e.g., inflammatory disorder) can comprise administering atherapeutically effective amount of IL-10 or an IL-10 delivery constructto an individual suffering from, suspected of suffering from, or inrelapse from the disease or condition (e.g., inflammatory disorder).

The term, “therapeutically effective amount,” as used herein, can meanthat the amount, e.g. of IL-10 or an IL-10 delivery construct, containedin a composition, e.g. formulation or oral formulation described herein,administered is of sufficient quantity to achieve the intended purpose,such as, for example, to treat a disease or condition, e.g. a disease orcondition causing inflammation. In some embodiments, administering aformulation to an individual comprises administering a therapeuticallyeffective amount of the formulation to the individual.

The individual in need thereof can be an individual refractory orresistant to at least one anti-inflammatory agent. The anti-inflammatoryagent can be an aminosalicylate. The aminosalicylate can be5-aminosalicylic acid (5-ASA; mesalazine), 4-amino salicylic acid(4-ASA), balsalazide, olsalazine, sulfasalazine, or a combinationthereof. The anti-inflammatory agent can be a corticosteroid. Thecorticosteroid can be an orally administered corticosteroid or anintravenously (IV) administered corticosteroid. The corticosteroid canbe prednisone. The anti-inflammatory agent can be an immunosuppressiveagent. The immunosuppressive agent can be azathioprine,6-mercaptopurine, or a combination thereof. The anti-inflammatory agentcan be a TNFα inhibitor. The TNFα inhibitor can be adalimumab,certolizumab, etanercept, golimumab, infliximab, or a combinationthereof. The at least one anti-inflammatory agent can be a Janus kinase(JAK) inhibitor. The JAK inhibitor can be filgotinib, upadacitinib,peficitinib, tofacitinib, or a combination thereof. The at least oneanti-inflammatory agent can be a sphingosine-1-phosphate (SIP) receptorantagonist. The SIP receptor antagonist can be ozanimod, amiselimod,etrasimod, or a combination thereof. The at least one anti-inflammatoryagent can be an integrin blocker. The integrin blocker can beetrolizumab, natalizumab, vedolizumab, abrilumab, carotegrast methyl, ora combination thereof. The at least one anti-inflammatory agent can bean IL-23 inhibitor. The IL-23 inhibitor can be ustekinumab, mirikizumab,brazikumab, guselkumab, risankizumab, or a combination thereof. The atleast one anti-inflammatory agent can be a phosphodiesterase 4 (PDE4)inhibitor. The at least one PDE4 inhibitor can be apremilast,cilomilast, roflumilast, tetomilast, rolipram, or a combination thereof.The at least one anti-inflammatory agent can be laquinimod.

In some cases, the individual in need thereof can be an individual whohas not been treated with an anti-inflammatory agent. In some cases, theindividual in need thereof can be an individual who has not been treatedwith 5-ASA. In some cases, the individual in need thereof can be anindividual who has responded partially or substantially to 5-ASA. Insome cases, the individual in need thereof may be treated with an IL-10delivery construct and 5-ASA.

In some embodiments, the oral formulations comprising an IL-10 deliveryconstruct described herein are orally administered to an individual inneed thereof. In some embodiments, the formulations comprising an IL-10delivery construct described herein are rectally administered to anindividual in need thereof. The formulations comprising the IL-10delivery construct can be administered to the individual for at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. The formulation canbe administered once a day, twice a day, or three times a day. Theindividual in need thereof can be a human. An individual in need thereofcan be an individual diagnosed with, suspected of having, or at risk ofa disease or condition causing inflammation. The disease or conditioncausing inflammation can be a disease or condition characterized by adeficiency in IL-10 expression. The disease or condition causinginflammation can be ulcerative colitis, inflammatory bowel disease(IBD), Celiac disease, proctitis, pouchitis, Crohn's disease, multiplesclerosis (MS), systemic lupus erythematosus (SLE), graft versus hostdisease (GVHD), rheumatoid arthritis, psoriatic arthritis, or psoriasis.The ulcerative colitis can be mild- to moderate ulcerative colitis ormoderate-to-severe ulcerative colitis. The Crohn's disease can befistulizing Crohn's disease. In some embodiments, the oral formulationis used in the treatment of the disorder causing inflammation. The oralformulation can be used to treat the disorder causing inflammation. Themethod can comprise administering a dose of an oral formulationcomprising IL-10 and one or more pharmaceutically acceptable excipientsto an individual. Administration of the dose of the oral formulation tothe individual can result in an immunomodulatory response.

The immunomodulatory response can comprise a decrease in a concentrationof fecal calprotectin (FCP) relative to an FCP baseline. Fecalcalprotectin is a biomarker of intestinal inflammation. Theconcentration of FCP can be determined from a fecal sample or from acolonic biopsy. The concentration of FCP can be determined by animmunoassay. The immunoassay can be an enzyme linked immunoassay(ELISA). In some embodiments, concentration of FCP is expressed in mg ofcalprotectin per kilogram of feces or μg of calprotectin per gram offeces. The decrease in the concentration of FCP can be a decrease of atleast 20%, 30%, 40%, or 50% relative to the FCP baseline. The decreasein the concentration of FCP can be a decrease of from about 50% to about80% relative to the FCP baseline. In some embodiments, the concentrationof FCP is decreased to 50 μg/g or less. In some embodiments, thedecrease in the concentration of FCP indicates a decrease ingastrointestinal inflammation.

The FCP baseline can be an initial concentration of FCP in an individualor population prior to the administration. The initial concentration ofFCP can be indicative of having a disease. An initial concentration ofFCP indicative of a disease can be an FCP concentration of greater than150 μg/g. In some embodiments, an FCP concentration of greater than 150μg/g is indicative of having ulcerative colitis (UC). In someembodiments, the concentration of FCP is decreased at least 50% relativeto the initial concentration of FCP and the dose of the oral formulationis from about 1 mg to about 3 mg.

The FCP baseline can be a placebo-adjusted FCP baseline. Theplacebo-adjusted FCP baseline can be a percent change of FCPconcentration following administration of a placebo to an individual orpopulation relative to initial FCP concentration prior to theadministration. In some embodiments, the concentration of FCP in anindividual or population treated with an IL-10 delivery construct isdecreased at least 20% relative to the placebo-adjusted FCP baseline andthe dose of the oral formulation of the IL-10 delivery construct is fromabout 1 mg to about 3 mg.

In one illustrative example, a placebo-administered individual orpopulation starts with an FCP concentration of 200 μg/g and increases to250 μg/g after the administration of a placebo (representing a 25%increase) and an IL-10 delivery construct administered individual orpopulation starts with an FCP concentration of 200 μg/g and decreases to100 μg/g after the administration of an IL-10 delivery construct(representing a 50% decrease). In this example, the FCP concentration ofthe IL-10 delivery construct administered individual or population canbe said to: (i) have a 50% reduction in FCP concentration when the FCPbaseline is an initial concentration of FCP in the IL-10 deliveryconstruct administered individual or population prior to theadministration, or (ii) have a 75% reduction (50%+25%) in FCPconcentration when the FCP baseline is the placebo-adjusted FCPbaseline.

In another illustrative example, a placebo administered individual orpopulation starts with an FCP concentration of 200 μg/g and decreases to150 μg/g after the administration of a placebo (representing a 25%decrease) and an IL-10 delivery construct administered individual orpopulation starts with an FCP concentration of 200 μg/g and decreases to100 μg/g after the administration of an IL-10 delivery construct(representing a 50% decrease). In this example, the FCP concentration ofthe IL-10 delivery construct administered individual or population canbe said to: (i) have a 50% reduction in FCP concentration when the FCPbaseline is an initial concentration of FCP in the IL-10 deliveryconstruct administered individual or population prior to theadministration, or (ii) have a 25% reduction (50%-25%) in FCPconcentration when the FCP baseline is the placebo-adjusted FCPbaseline.

The immunomodulatory response can comprise a decrease in a concentrationof C-Reactive Protein (CRP) relative to a CRP baseline. C-ReactiveProtein (CRP) is a biomarker of systemic inflammation. The concentrationof CRP can be determined from a blood sample. The CRP concentration canbe a serum CRP concentration. The concentration of CRP can be determinedby an immunoassay or a nephelometric assay. The immunoassay can be anenzyme linked immunoassay (ELISA). The decrease in the concentration ofCRP can be a decrease of at least at least 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, or 80% relative to the CRP baseline. The decrease in theconcentration of CRP can be a decrease of from about 5% to about 60%,from about 10% to about 50%, from about 30% to about 90%, or from about40% to about 80% relative to the CRP baseline. In some embodiments, theconcentration of CRP is decreased to less than 5 mg/L. In someembodiments, the decrease in the concentration of CRP indicates adecrease in systemic or gastrointestinal inflammation.

The CRP baseline can be an initial concentration of CRP in an individualor population prior to the administration. The initial concentration ofCRP can be indicative of having a disease. An initial concentration ofCRP indicative of a disease can be a CRP concentration of greater than 5mg/L. In some embodiments, a CRP concentration of greater than 5 mg/L isindicative of irritable bowel disease (IBD). In some embodiments, theconcentration of CRP is decreased at least 40% relative to the initialconcentration CRP and the dose of the oral formulation is from about 1mg to about 3 mg.

The CRP baseline can be a placebo-adjusted CRP baseline. Theplacebo-adjusted CRP baseline can be a percent change of CRPconcentration following administration of a placebo to a placeboadministered individual or population relative to initial CRPconcentration prior to the administration. In some embodiments, theconcentration of CRP in an individual or population treated with anIL-10 delivery construct is decreased at least 10% relative to theplacebo-adjusted CRP baseline and the dose of the oral formulation ofthe IL-10 delivery construct is about 3 mg. In some embodiments, theconcentration of CRP in an individual or population treated with anIL-10 delivery construct is decreased at least 40% relative to theplacebo-adjusted CRP baseline and the dose of the oral formulation ofthe IL-10 delivery construct is about 1 mg.

In one illustrative example, a placebo-administered individual orpopulation starts with a CRP concentration of 8 mg/L and increases to 10mg/L after the administration of a placebo (representing a 25% increase)and an IL-10 delivery construct-administered individual or populationstarts with a CRP concentration of 8 mg/L and decreases to 4 mg/L afterthe administration of an IL-10 delivery construct (representing a 50%decrease). In this example, the CRP concentration of the IL-10 deliveryconstruct administered individual or population can be said to: (i) havea 50% reduction in CRP concentration when the CRP baseline is an initialconcentration of CRP in the IL-10 delivery construct-administeredindividual or population prior to the administration, or (ii) have a 75%reduction (50%+25%) in CRP concentration when the CRP baseline is theplacebo-adjusted CRP baseline.

In another illustrative example, a placebo-administered individual orpopulation starts with a CRP concentration of 8 mg/L and decreases to 6mg/L after the administration of a placebo (representing a 25% decrease)and an IL-10 delivery construct-administered individual or populationstarts with a CRP concentration of 8 mg/L and decreases to 4 mg/L afterthe administration of an IL-10 delivery construct (representing a 50%decrease). In this example, the CRP concentration of the IL-10 deliveryconstruct administered individual or population can be said to: (i) havea 50% reduction in CRP concentration when the CRP baseline is an initialconcentration of CRP in the IL-10 delivery construct administeredindividual or population prior to the administration, or (ii) have a 25%reduction (50%-25%) in CRP concentration when the CRP baseline is theplacebo-adjusted CRP baseline.

The immunomodulatory response can comprise a decrease in a Geboes scorerelative to a Geboes score baseline. Geboes scoring system is a standardmeasure of histological response (Geboes et al. Gut. 2000 September;47(3):404-9). As used herein, a Geboes score can be a 0-22 pointhistologic scoring system in which higher scores represent more severedisease. The Geboes score baseline can be an initial Geboes score of anindividual or population prior to the administration. The Geboes scorebaseline can be a placebo-adjusted Geboes score baseline. Theplacebo-adjusted Geboes score baseline can be a difference of a Geboesscore following administration of a placebo to an individual orpopulation from an initial Geboes score prior to administration of theplacebo. In some embodiments, the Geboes score is decreased a least 2units relative to the placebo-adjusted Geboes score baseline and thedose of the oral formulation is from about 1 mg to about 30 mg.

In one illustrative example, a placebo-administered individual orpopulation starts with a Geboes score of 10, which increases to 12following administration of a placebo (representing an increase of 2units or 20%) and an IL-10 delivery construct individual or populationstarts with a Geboes score of 10, which decreases to 5 followingadministration of an IL-10 delivery construct (representing a decreaseof 5 units or 50%). In this example the Geboes score of the IL-10delivery construct administered individual can be said to: (i) have adecrease of 5 units or 50% when the Geboes score baseline is the initialGeboes score of the IL-10 delivery construct administered individual, or(ii) have a decrease of 7 units or 70% (50%+20%) when the Geboes scorebaseline is the placebo-adjusted Geboes score baseline.

In another illustrative example, a placebo-administered individual orpopulation starts with a Geboes score of 10, which decreases to 8following administration of a placebo (representing an decrease of 2units or 20%) and an IL-10 delivery construct individual or populationstarts with a Geboes score of 10, which decreases to 5 followingadministration of an IL-10 delivery construct (representing a decreaseof 5 units or 50%). In this example the Geboes score of the diseasedindividual can be said to: (i) have a decrease of 5 units or 50% whenthe Geboes score baseline is the initial Geboes score of the IL-10delivery construct administered individual, or (ii) have a decrease of 3units or 30% (50%-20%) when the Geboes score baseline is theplacebo-adjusted Geboes score baseline.

In some embodiments, the immunomodulatory response comprises a decreasein a concentration of FCP relative to an FCP baseline and a decrease ina concentration of CRP relative to a CRP baseline. In some embodiments,the immunomodulatory response comprises a decrease in a concentration ofFCP relative to an FCP baseline and a decrease in a Geboes scorerelative to a Geboes score baseline. In some embodiments, theimmunomodulatory response comprises a decrease in a concentration of CRPrelative to a CRP baseline and a decrease in a Geboes score relative toa Geboes score baseline. In some embodiments, the immunomodulatoryresponse comprises a decrease in a concentration of FCP relative to anFCP baseline, a decrease in a concentration of CRP relative to a CRPbaseline, and a decrease in a Geboes score relative to a Geboes scorebaseline

Colonic tissue of an individual with a gastrointestinal inflammatorydisorder can show infiltration of the lamina propria by mononuclearcells, eosinophils, and histiocytes, or a combination thereof inaddition to neutrophilic infiltration into the epithelium associatedwith crypt architecture destruction, erosions, and ulcerations.Administration of an IL-10 delivery construct to an individual canresults in a reduction in the infiltration of the lamina propria bymononuclear cells, eosinophils, and histiocytes, or a combinationthereof in addition to neutrophilic infiltration into the epitheliumassociated with crypt architecture destruction, erosions, andulcerations.

In some embodiments, less than 5%, less than 4%, less than 3%, less than2%, or less than 1% of the IL-10 of the IL-10 delivery construct entersthe bloodstream.

In some embodiments, the oral formulations comprising an IL-10 deliveryconstruct described herein are orally administered to an individual inneed thereof. The formulations comprising the IL-10 delivery constructcan be administered to the individual for at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, or 14 days. The formulation can be administeredonce a day.

Administration of a therapeutically effective amount of a dose of anoral formulation from about 1 mg to about 60 mg to an individual canresult in a greater than 20% increase in a plasma concentration ofIL-1Ra in the individual relative to a baseline plasma concentration ofIL-1Ra. In some embodiments, the baseline plasma concentration of IL-1Rais a plasma concentration of IL-1Ra of the individual prior to theadministration. In some embodiments, the dose of the oral formulation isfrom about 3 mg to about 30 mg and the increase in the plasmaconcentration of IL-1Ra relative to the baseline plasma concentration ofIL-1Ra is greater than 30%. In some embodiments, the dose of the oralformulation is from about 3 mg to about 30 mg and the increase in theplasma concentration of IL-1Ra relative to the baseline plasmaconcentration of IL-1Ra is from 30% to 45%. In some embodiments, thedose of the oral formulation is from about 3 mg to about 10 mg and theincrease in the plasma concentration of IL-1Ra relative to the baselineplasma concentration of IL-1Ra is from 30% to 35%. In some embodiments,the dose of the oral formulation is about 30 mg and the increase in theplasma concentration of IL-1Ra relative to the baseline plasmaconcentration of IL-1Ra is from 40% to 43%. The dose of the oralformulation can refer to the amount of IL-10 or an IL-10 deliveryconstruct in the oral formulation.

Administration of the dose of oral formulation to the individual canresult in a plasma concentration of IL-10 in the individual that doesnot exceed 1500 μg/mL, 1000 μg/mL, 900 μg/mL, 800 μg/mL, 700 μg/mL, 600μg/mL, 500 μg/mL, 400 μg/mL, 300 μg/mL, 200 μg/mL, 100 μg/mL, 50 μg/mL,or 10 μg/mL. Administration of the dose of oral formulation to theindividual can result in a plasma concentration of IL-10 in theindividual that does not exceed 1500 μg/mL. Administration of the doseof oral formulation to the individual can result in a plasmaconcentration of IL-10 in the individual that does not exceed 1000μg/mL. Administration of the dose of oral formulation to the individualcan result in a plasma concentration of IL-10 in the individual thatdoes not exceed 500 μg/mL. Administration of the dose of oralformulation to the individual can result in a plasma concentration ofIL-10 in the individual that does not exceed 100 μg/mL.

Administration of a therapeutically effective amount of a formulationcomprising IL-10 to an individual can result in an increase in aconcentration of IL-1Ra in plasma of the individual can result in anincrease in a concentration of IL-1Ra in plasma of the individual of atleast 5000 μg/mL relative to a baseline level of IL-1Ra. The baselinelevel of IL-1Ra can be a typical concentration of IL-1Ra in the plasmaof the individual prior to the administration. The concentration ofIL-1Ra can reach a maximum of at least 5000 μg/mL, 6000 μg/mL, 7000μg/mL, 8000 μg/mL, 9000 μg/mL, 10,000 μg/mL, 11,000 μg/mL, 12,000 μg/mL,15,000 μg/mL, 20,000 μg/mL, or 25,000 μg/mL. The concentration of IL-1Racan reach a maximum of from 1000 μg/mL to 10,000 μg/mL, from 8000 μg/mLto 12,000 μg/mL, or from 25,000 μg/mL to 28,000 μg/mL. The maximumconcentration of IL-1Ra can be reached after at least 1, 2, 3, or 4hours. The maximum concentration of IL-1Ra can be reached from 2 to 4hours, from 2 to 3 hours, or from 3 to 4 hours after the administration.In one example, a concentration of IL-1Ra can reach a maximum of from25,000 μg/mL to 28,000 μg/mL at from 2.5 hours to 3.5 hours postadministration. In another example, a concentration of IL-1Ra can reacha maximum of from 8,000 μg/mL to 12,000 μg/mL at from 3.5 hours to 4.5hours post administration.

Administration of a therapeutically effective amount of a formulationcomprising IL-10 to an individual with a disease or condition (e.g.,inflammatory disorder) can result in at least one of: (1) a peak IL-10concentration of less than 50 μg/mL in the plasma of the individual and(2) co-localization of the IL-10 with a cell expressing CD3 in a laminapropria of the individual. The peak IL-10 concentration can be less than50 μg/mL, 40 μg/mL, 30 μg/mL, 20 μg/mL, 10 μg/mL, 2.5 μg/mL, 2.0 μg/mL,1.5 μg/mL, or 1.0 μg/mL. The peak IL-10 concentration can be reachedfrom 1 hour to 5 hours, from 1 hour to 3 hours, from 2 hours to 3 hours,or from 3 hours to 5 hours after the administration. The cell expressingCD3 can be a lymphocyte. The lymphocyte can be a T cell.

Administration of a therapeutically effective amount of a formulationcomprising IL-10 to an individual with a disease or condition (e.g.,inflammatory disorder) can result in an increase in a ratio ofexpression of IL-Ra to interleukin 1 beta (IL-1β) (IL-1Ra:IL-1β) in thecolonic tissue of the individual. The ratio of IL-1Ra:IL-1β can be atleast 1:1, 1.5:1, 2:1, 2.5:1, or 3:1. The ratio of IL-1Ra:IL-1β can befrom 2:1 to 3:1.

Administration of a therapeutically effective amount of a formulationcomprising IL-10 to an individual with a disease or condition (e.g.,inflammatory disorder) can result in no significant increase in aconcentration of at least one pro-inflammatory cytokine in plasma of theindividual. The at least one pro-inflammatory cytokine can be interferongamma (IFN-γ), IL-1β, interleukin 2 (IL-2), interleukin 8 (IL-8), or acombination thereof. Administration of a therapeutically effectiveamount of a formulation comprising IL-10 to an individual with a diseaseor condition (e.g., inflammatory disorder) can result in an increase ina concentration of IL-1Ra in plasma of the individual. The concentrationof IL-1Ra can reach a maximum of from 25,000 pg/mL to 28,000 pg/mL atfrom 2.5 hours to 3.5 hours post administration.

Administration of a therapeutically effective amount of a formulationcomprising IL-10 to an individual with a disease or condition (e.g.,inflammatory disorder) can result in: (a) an increase in expression ofinterleukin 1 receptor agonist (IL-1Ra) in a colonic tissue of theindividual; (b) a decrease in expression of at least onepro-inflammatory gene in the colonic tissue; (c) an increase inexpression of at least one anti-inflammatory gene in the colonic tissueof the individual; (d) an increase in expression of at least one tissuerepair gene in the colonic tissue of the individual; (e) an increase inexpression of at least one anti-microbial gene in the colonic tissue ofthe individual; or (f) a combination thereof. In some embodiments,administration of a therapeutically effective amount of a formulationcomprising IL-10 to an individual with a disease or condition (e.g.,inflammatory disorder) results in an increase in a ratio of expressionof Il-1Ra to interleukin 1 beta (IL-1R: IL-10) in the colonic tissue ofthe individual. The ratio of IL-1R: IL-10 can be greater than 1:1,1.5:1, 2:1, 2.5:1, or 3:1.

The at least one pro-inflammatory gene can be MHC-II, HPGDS, FCER1A,PLA2G2D, CCL13, FUT3, CCL28, UGT1A1, CCL20, NLRP1, TPH, or a combinationthereof. The at least one anti-inflammatory gene can be CD163, SCNN1G,STC1, HGF, SGK1, miR-24-2, SCNN1B, PTGDR, MTNR1A, ACE2, NOX2, BEST2,VNN2, LTB4R2, B2GALT5, or a combination thereof. The at least one tissuerepair gene can be SCNN1G, STC1, TIMP1, SCNN1B, BEST2, B3GALT5, or acombination thereof. The at least anti-microbial gene can be PI15, PI3,BDKRB1, CC128, SERPINE2, or a combination thereof.

Further described herein, in certain embodiments, are methods ofpreventing a recurrence of an inflammatory disorder in an individual inremission for the inflammatory disorder, comprising administering aformulation comprising IL-10 and one or more pharmaceutically acceptableexcipients to the individual. The individual can have been in remissionfor the inflammatory disorder for at least one month, 6 months, 8months, 1 year, 2 years, 3 years, 4 years, or 5 years.

Described herein, in certain embodiments, are methods of treating aninflammatory disorder in an individual refractory or resistant to atleast one anti-inflammatory agent, the method comprising administering aformulation comprising IL-10 to the individual. The anti-inflammatoryagent can be an aminosalicylate. In some embodiments, theaminosalicylate is selected from the group consisting of5-aminosalicylic acid (5-ASA; mesalazine), 4-amino salicylic acid(4-ASA), balsalazide, olsalazine, and sulfasalazine. Theanti-inflammatory agent can be a corticosteroid. In some embodiments,the corticosteroid is prednisone. In some embodiments, thecorticosteroid is an orally administered corticosteroid or anintravenously (IV) administered corticosteroid. The anti-inflammatoryagent can be an immunosuppressive agent. In some embodiments, theimmunosuppressive agent is selected from the group consisting ofazathioprine, 6-mercaptopurine, and a combination thereof. Theanti-inflammatory agent can be a TNFα inhibitor. In some embodiments,the TNFα inhibitor is selected from the group consisting of adalimumab,certolizumab, etanercept, golimumab, and infliximab. The at least oneanti-inflammatory agent can be a Janus kinase (JAK) inhibitor. In someembodiments, the JAK inhibitor is selected from the group consisting offilgotinib, upadacitinib, peficitinib, and tofacitinib. The at least oneanti-inflammatory agent can be a sphingosine-T-phosphate (SIP) receptorantagonist. In some embodiments, the SIP receptor antagonist is selectedfrom the group consisting of ozanimod, amiselimod, and etrasimod. The atleast one anti-inflammatory agent can be an integrin blocker. In someembodiments, the integrin blocker is selected from the group consistingof etrolizumab, natalizumab, vedolizumab, abrilumab, and carotegrastmethyl. The at least one anti-inflammatory agent can be an IL-23inhibitor. In some embodiments, the IL-23 inhibitor is selected from thegroup consisting of ustekinumab. mirikizumab, brazikumab, guselkumab,and risankizumab. The at least one anti-inflammatory agent can be aphosphodiesterase 4 (PDE4) inhibitor. In some embodiments, the at leastone PDE4 inhibitor is selected from the group consisting of apremilast,cilomilast, roflumilast, tetomilast, and rolipram. The at least oneanti-inflammatory agent can be laquinimod. In some embodiments, theindividual is administered the formulation daily for at least 5, 7, 10,12, or 14 days.

Combination Therapies

Provided herein are methods of treating an inflammatory disease in asubject in need thereof, comprising administering an IL-10 therapeuticin combination with a non-IL-10 immunosuppressor. In some cases, theIL-10 therapeutic is an oral therapeutic. In some cases, treatment withthe non-IL-10 immunosuppressor is commenced prior to treatment with theoral IL-10 therapeutic. In some cases, treatment with the non-IL-10immunosuppressor is commenced concomitantly with treatment with the oralIL-10 therapeutic. In some cases, the subject has previously beentreated with a non-IL-10 immunosuppressor and had an inadequateresponse. In some cases, the subject is predicted to respondinadequately to a non-IL-10 immunosuppressor based on medical history,family history, genetics, or expression of biomarkers. In some cases,the non-IL-10 immunosuppressor is not an interleukin.

In some cases, the non-IL-10 immunosuppressor is an anti-integrintherapy, for example vedolizumab. In some cases, the non-IL-10immunosuppressor is a Janus kinase inhibitor (JAK inhibitor). In somecases, the non-IL-10 immunosuppressor is an IL-23 antagonist and/or anIL-12/IL-23 antagonist. In some cases, the non-IL-10 immunosuppressor isa Sphingosine-T-phosphate (STP) modulator or a Sphingosine-T-phosphatereceptor modulator. In other cases, the non-IL-10 immunosuppressor isIL-22 or an IL-22 agonist.

In some cases, the non-IL-10 immunosuppressor is a TNF alpha inhibitor.In some cases, treatment with the TNF alpha inhibitor is commenced priorto treatment with the IL-10 therapeutic. In some cases, treatment withthe TNF alpha inhibitor is commenced concomitantly with treatment withthe IL-10 therapeutic. In some cases, the subject has previously beentreated with a TNF alpha inhibitor and had an inadequate response. Insome cases, the subject is predicted to respond inadequately to a TNFalpha inhibitor based on medical history, family history, genetics, orexpression of biomarkers.

Further provided are methods of treating an inflammatory disease in asubject, wherein the subject has had an inadequate response to treatmentwith a TNF alpha inhibitor, the method comprising administering an IL-10therapeutic. In some cases, treatment with the TNF alpha inhibitor iscontinued concomitantly with the IL-10 therapeutic.

In some cases, the inflammatory disease may be a disease of theintestines or digestive tract. In some cases, the inflammatory diseasemay manifest or present in a tissue distal to or at a remote distancefrom the digestive tract. In some cases, the inflammatory disease may beselected from the group consisting of: inflammatory bowel disease,psoriasis, plaque psoriasis, hidradenitis suppurativa, psoriaticarthritis, rheumatoid arthritis, juvenile idiopathic arthritis,ankylosing spondylitis, bacterial sepsis, Crohn's disease, fistulizingCrohn's disease, moderate-to-severe ulcerative colitis, mild-to-moderateulcerative colitis, ulcerative colitis, collagenous colitis, lymphocyticcolitis, ischaemic colitis, diversion colitis, Behcet's syndrome,indeterminate colitis, rheumatoid arthritis, pancreatitis, liverinflammation, pouchitis, proctitis, uveitis, graft vs host disease, andepithelial cell injury. In some cases, the inflammatory disease isrheumatoid arthritis. In some cases, the inflammatory disease is aninflammatory bowel disease. In some cases, the inflammatory boweldisease is ulcerative colitis. In some cases, the inflammatory boweldisease is Crohn's disease.

In some cases, an IL-10 therapeutic may be administered locally to asite of disease. For example, an IL-10 therapeutic may be administeredorally to treat a disease of the digestive tract such as ulcerativecolitis or Crohn's disease. In some cases, the IL-10 therapeutic may beadministered orally to achieve a systemic dose which may treat a diseasedistal to the digestive tract. For example, an IL-10 therapeutic may beadministered orally to treat rheumatoid arthritis or psoriasis.

An inadequate response to a therapeutic may comprise a partial response,or a lack of response. In some cases, an inadequate response is aresponse other than a complete cure or complete remission of a disease.A subject who has had an inadequate response to a therapeutic may havefewer symptoms or may have less severe symptoms during or after thetreatment as compared to prior to the treatment. In other cases, asubject who has had an inadequate response to a disease may have thesame number of symptoms or the same symptoms as prior to treatment. Insome cases, a subject who has had an inadequate response to atherapeutic may have more symptoms or more severe symptoms aftertreatment as compared to prior to treatment.

In some cases, a subject with rheumatoid arthritis who has had aninadequate response to a therapeutic may continue to have one or moresymptoms of arthritis after treatment with the therapeutic (e.g., ananti-TNF alpha inhibitor alone). For example, the subject may have oneor more joints with active disease. Active disease may be identified byfluorescent optical imaging or magnetic resonance imaging. The subjectwith an inadequate response to a rheumatoid arthritis treatment may haveone or more joints which are tender, and/or one or more joints which areswollen. Other symptoms which may be present include stiffness orweakness of joints, redness of the skin over joints, lumps over thejoints, flare, dry mouth, physical deformity, or a sensation of pins andneedles.

In some cases, a subject with ulcerative colitis who has had aninadequate response to a therapeutic may continue to have one or moresymptoms of ulcerative colitis after treatment with the therapeutic(e.g., an anti-TNF alpha inhibitor alone). The subject with aninadequate response to an ulcerative colitis treatment may have amodified Mayo Clinic Score (MMS) of between about 4 points and about 9points. The subject may have a centrally read MCS endoscopic sub scoreof grade 2 or higher. In some cases, the subject may have a MMS rectalbleeding sub score of 1 point or higher. In some cases, the subject mayhave disease extending 15 cm or more from the anal verge. Other symptomsof ulcerative colitis include abdominal pain/discomfort, blood or pus instool, fever, weight loss, frequent recurring diarrhea, fatigue, reducedappetite, and tenesmus.

In some cases, a subject may have had an inadequate response aftertreatment with a TNF alpha inhibitor. In some cases, the subject mayhave had an inadequate response to the TNF alpha inhibitor after atleast 6 or at least 12 weeks of treatment with the TNF alpha inhibitor.

The TNF alpha inhibitor may be a monoclonal antibody. Examples ofanti-TNF alpha therapeutics include of infliximab (Remicade), adalimumab(Humira) and golimumab (Simponi). In some cases, the TNF alpha inhibitoris etanercept. In some cases, the TNF alpha inhibitor is not etanercept.

The TNF alpha inhibitor may be administered by subcutaneous injection,or by any other suitable method. For example, adalimumab may beadministered by subcutaneous injection. In some cases, adalimumab may beadministered at a dose of 40 mg every other week. In some cases, one ormore initial doses may be higher than a maintenance dose. For example,an adalimumab therapy regimen may comprise a first dose of 160 mg,followed by a second dose of 80 mg about two weeks later, followed twoweeks later by maintenance doses of 40 mg every other week. In somecases, an initial dose of adalimumab may be 80 mg, followed two weekslater by maintenance doses of 40 mg every other week.

The TNF alpha inhibitor may be administered by intravenous infusion. Forexample, infliximab may be administered by intravenous infusion. In somecases, the TNF alpha inhibitor may be administered by intravenousinfusion over a period of time of at least two hours. In some cases,infliximab may be administered at a dose of 5 mg/kg. In other cases,infliximab may be administered at a dose of 3 mg/kg, or at a dose of 10mg/kg. In some cases, a treatment regimen may comprise more frequentinitial doses, followed by maintenance doses. In some cases, a treatmentregimen may comprise administering the TNF alpha inhibitor at 0, 2, and6 weeks, then every 8 weeks.

The TNF alpha inhibitor may be administered by a medical professional ormay be provided to a patient for self-administration. In some cases, theTNF alpha inhibitor may be provided in a single-dose prefilled syringe,or in a single dose automatic injector. For example, adalimumab may beprovided in a single-dose HUMIRA Pen.

In some cases, an IL-10 therapeutic may be administered orally. In somecases, an IL-10 therapeutic may be administered approximatelysimultaneously with a TNF alpha inhibitor. For example, an IL-10therapeutic may be administered immediately before, or immediately aftera TNF alpha inhibitor. In some cases, an IL-10 therapeutic may beadministered on the same day as a TNF alpha inhibitor, or on the dayproceeding or day following administration of a TNF alpha inhibitor. Forexample, an IL-10 therapeutic and a TNF alpha inhibitor may beadministered at 0, 2, and 6 weeks, and then subsequently every 8 weeks.In another example, an IL-10 therapeutic and a TNF alpha inhibitor maybe administered every two weeks.

In some cases, an IL-10 therapeutic may be an IL-10 delivery construct.

EXAMPLES Example 1: IL-10 Delivery Construct Design

IL-10 is an immunomodulatory cytokine that suppresses the activation andeffector function of multiple innate and adaptive immune cells. An IL-10delivery construct (SEQ ID NO: 5) was designed. This construct was arecombinant, homodimeric fusion protein where each monomer consisted ofan N-terminal methionine, a cholix³⁸⁶ domain (SEQ ID NO: 4) and arecombinant human IL-10 (rhIL-10) domain (SEQ ID NO: 2) connected by anamino acid polypeptide spacer of glycine and serine (polyGlySer)residues (SEQ ID NO: 6). The cholix³⁸⁶ domain was a truncated form of avariant of cholix, a non-toxic mutant derived from Vibrio choleracontaining 386 amino acids. The construct had a molecular weight of125,796 Da, and an isoelectric point (pI) of 5.49.

The cholix³⁸⁶ domain facilitates active transport of the IL-10 deliveryconstruct of SEQ ID NO: 5 across epithelial cells via vesiculartranscytosis to the local gastrointestinal submucosal tissue. Targeteddelivery of the rhIL-10 directly to the lamina propria via the oralroute may bypass one or more drawbacks experienced with systemicadministration and translate into higher mucosal concentrations andclinically meaningful reductions in inflammation and disease.

As used in the examples herein, Drug Substance (DS) was used whenreferring to the lyophilized powder and Drug Product (DP) was used whenreferring to the capsule or tablet form.

Example 2: Expression of the Target Construct

Plasmids containing the coding sequence (SEQ ID NO: 10) of the targetconstruct (SEQ ID NO: 5) were constructed by cloning into the Nde I andEcoR I sites of a pET26(b) backbone. The sequence encoded by SEQ ID NO:10 is a codon-improved sequence for expression in bacterial cells. Theplasmid contained the T7 promoter and conferred kanamycin resistance.BL21 E. coli cells were transfected with the target construct plasmidusing a heat shock method of transformation as follows: BL21 cells andthe target construct plasmid were aliquoted into a tube and incubated onice for 30 minutes. The tubes were then heat shocked for 30 to 45seconds at 42° C.±2° C. in a water bath. Immediately after the heatshock, the tubes were placed in ice for 2 to 5 minutes. Media was addedinto each tube and the tubes were incubated for 60 minutes at 37° C. Thetransformed cells were plated onto LB/Kan agar plates and incubatedovernight at 37° C. A single colony was then picked from the agar plate,inoculated in 4 mL of LB media, and grown overnight in a shaker flask.Glycerol stock (80%) was added to the culture, which was then filledinto cryovials and stored at −80±10° C. The pre-RCB (Research Cell Bank;the culture+glycerol stock) was then further manufactured to produce themaster cell bank (MCB).

To produce the MCB, cells from the RCB were expanded in shake flasksuntil sufficient cell mass was accumulated, recovered by centrifugation,resuspended in cryopreservation medium, aliquoted into 300 cryovials,and cooled until frozen. The MCB was stored at −80±5° C. in a controlledaccess GMP facility. The MCB was manufactured and is maintained inaccordance with cGMP procedures and ICH Guidelines Q5B and Q5D.

Following expansion in a shake flask, cells were transferred into aproduction bioreactor. Fermentation was executed in a 1500 L bioreactorin the presence of kanamycin for selective pressure. Productionfermentation consists of a cell growth phase followed by an expressionphase using isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer,where the protein was expressed intracellularly as insoluble inclusionbodies. The production reactor was controlled at set pH, temperature,and dissolved oxygen level as specified in the manufacturing procedure:pH was controlled by phosphoric acid and ammonium hydroxide addition;dissolved oxygen was controlled by air and oxygen gas flows. All gaseswere passed through membrane filters of pore size 0.22 μm or less. Theproduction reactor contained bacterial growth medium with definedcomponents. Before inoculation, these ingredients were sterilizedaccording to written standard operating procedures. The production phasewas a fed batch process, where glucose-based feed media are added tomaintain cell growth and culture viability.

At the end of production, the cells were harvested by centrifugation,and the cell paste processed further or frozen for processing at a laterdate. High-pressure homogenization was used to release theproduct-containing inclusion bodies. The inclusion bodies were thenresuspended, washed, and separated from other cellular components bycentrifugation. The inclusion bodies were either processed forwardimmediately or frozen for use at a later date. The cell paste and/orinclusion body slurry was stored at −20±5° C. prior to further use.

Example 3: Refolding Optimization

Solubilization of inclusion bodies (IBs) was carried out using a highconcentration of guanidine hydrochloride, a strong chaotrope. Followingsolubilization, an initial effort using a traditional refolding approachreducing with DTT and then diluting into a redox cocktail generated alow yield of properly folded dimer (<5% dimer). A second approachutilizing a sulfitolysis generated a higher recovery and wasimplemented. The peptide was first reduced with sodium sulfite and thenthe free sulfhydryls were capped with potassium tetrathionate. Followingdiafiltration to remove the residual sulfitolysis reagents, the proteinwas diluted into a redox cocktail, which allowed the protein to refoldand oxidize. Utilizing this approach, the yield was ˜2-fold higher (˜10%dimer) but still lower than desired. The effect of refolding in thepresence of osmolytes, such as sucrose as well as other water modifyingagents such as glycerol, was further investigated.

Refolding efficiency was assessed after varying arginine concentrationand the target construct (protein) concentration of the refoldingsolution (TABLE 5; FIGS. 25A-25B). The refolding efficiency shown in thetables and figures in this example is equivalent to the percent dimersof the resulting refolded target constructs and was assessed by sizeexclusion high performance liquid chromatography (SE-HPLC). An examplechromatogram showing dimer % from use of four different refoldingsolutions is shown in FIG. 28. The refolding efficiencies of the fourrefolding solutions illustrated in the chromatogram of FIG. 28 are shownin FIG. 29.

TABLE 5 Concentrations of components in nine refolding solutions varyingarginine (M) and target construct concentrations (mg/mL) ArginineProtein Conc 1 0.50 0.75 2 0.50 1.00 3 0.50 1.50 4 0.75 1.00 5 0.75 1.006 0.75 1.50 7 1.00 0.75 8 1.00 1 9 1.00 1.5

Refolding efficiency was assessed after varying the pH and glycerolconcentration of the refolding solution (TABLE 6; FIGS. 26A-26B).

TABLE 6 Concentrations of components in nine refolding solutions varyingglycerol (mM) concentration and pH Protein Arginine Conc Glycerol pH 10.75 1.00 0 7.5 2 0.75 1.00 10 7.5 3 0.75 1.00 30 7.5 4 0.75 1.00 0 8 50.75 1.00 10 8 6 0.75 1.00 30 8 7 0.75 1.00 0 8.5 8 0.75 1.00 10 8.5 90.75 1.00 30 8.5

Refolding efficiency was assessed after varying sucrose concentrationand PEG concentration of the refolding solution (TABLE 7; FIGS.27A-27B).

TABLE 7 Concentrations of components in nine refolding solutions varying% PEG and sucrose concentrations (M) Arginine Protein Conc PEG 3350Sucrose, M 1 0.75 1.00 0 0 2 0.75 1.00 0.1 0 3 0.75 1.00 0.5 0 4 0.751.00 0 0.25 5 0.75 1.00 0.1 0.25 6 0.75 1.00 0.5 0.25 7 0.75 1.00 0 0.58 0.75 1.00 0.1 0.5 9 0.75 1.00 0.5 0.5

Refolding efficiency was assessed after varying sucrose, glycerol, andPEG concentration of the refolding solution (TABLE 8).

TABLE 8 Concentrations of components in ten refolding solution andresulting refolding efficiency (dimer %) Arginine, Protein Conc,Sucrose, Glycerol, PEG Dimer M mg/ml M % 3350, % (%) 1 1.00 1.00 0 0 0.217.38% 2 1.00 1.00 0.1 0 0 18.29% 3 1.00 1.00 0.25 0 0 18.16% 4 1.001.00 0 5 0.1 17.71% 5 1.00 1.00 0.1 5 0.2 19.00% 6 1.00 1.00 0.25 5 0.118.21% 7 1.00 1.00 0 10 0 16.30% 8 1.00 1.00 0.1 10 0.1 18.10% 9 1.001.00 0.25 10 0.2 16.61% 10 1.00 1.00 0.25 5 0 17.90%

Example 4: Purification of Refolded Constructs

Several modes of chromatography were evaluated during processdevelopment. Cation exchange (CEX) was unsuccessful at relatively low pHas the protein would precipitate at a pH below the pI of the targetconstruct (pH 5.5). Hydrophobic Interaction Chromatography (HIC) wasalso unsuccessful, as the protein appeared to be unstable in the highsalt necessary for binding.

Anion exchange (AEX) worked well, as the protein was stable at higher pHand bound at reasonable capacity. Several AEX supports from variousvendors were evaluated, with the Capto™ Q ImpRes giving the best overallperformance, particularly with respect to the separation of the activedimer species from the two major product-related impurities, residualmonomer and aggregated species. As a polishing step, ceramichydroxyapatite (CHT) was implemented as a mixed-mode orthogonal step tofurther reduce product and process related impurities.

Refolded target constructs were subjected to AEX chromatograph followedby CHT chromatography. Gradient elutions on both chromatography stepswere utilized for the initial clinical campaign, with the opportunity todevelop optimized step elutions being evaluated as clinical developmentprogresses. During elution, fractions were collected and each fractionassayed by SE-HPLC for dimer content of the target construct. Fractionscontaining above a specified threshold (e.g., 75%) were then pooled inorder to meet the desired dimer content percentage. Following the CHTstep, the final bulk was concentrated and diafiltered using UF/DF intothe formulation buffer. SDS-PAGE analysis of the major processintermediates is shown in FIG. 30, which demonstrated an increase indimer purity during downstream processing.

Example 5: Lyophilization of the Liquid Intermediate

In order to generate an oral capsule containing the IL-10 deliveryconstruct, the purified liquid intermediate produced following thepurification protocol described in Example 4, was transformed into adried powder. Lyophilization was determined to be an appropriate way toproduce the powder while minimizing aggregate formation.

Formulation development to establish the lyophilization buffer wasperformed by initially screening the freeze-thaw and shear-inducedliquid stability of the target construct following dialysis intocombinations of several components outlined below:

1) Surfactants: Polysorbate 80, 20 and Poloxamer 188

2) pH range from 5 to 83) Osmolyte: 5% sucrose4) Salts: Sodium chloride (NaCl), potassium chloride (KCl), magnesiumchloride (MgCl₂), ammonium sulfate (NH₄SO₄) and sodium sulfate (Na₂SO₄).

Based on the data from the initial liquid formulation screen it wasdetermined that poloxamer 188 reduced aggregation of the targetconstruct under shear stress, and that phosphate buffered saline (PBS)and NaCl at 150-200 mM also demonstrated increased stability.

Further testing based on the results from the initial liquid formulationscreen, initial lyophilization feasibility studies were conducted usingthe conditions in TABLE 9. Glycine and mannitol, known to be useful inthe lyophilization of proteins as amorphous bulking agents were added tothis screen, histidine at pH 7.0 added to provide a more granularevaluation of the effect of pH in the 7.0-7.5 range, and trehalose wasadded as an option to sucrose as an osmolyte. FIGS. 21 and 22 show thepercentage of target constructs in the dimer form before and afterincubation at 25° C. for 3 days (FIG. 21) and before and after 5freeze/thaw cycles (FIG. 22).

TABLE 9 Summary of conditions used in initial lyophilization feasibilitystudies Buffer Bulking Agent Stabilizer 10 mM Histidine pH — 150 mM NaCl7.0 2% Glycine — 2% Glycine 0.5% Sucrose 2% Glycine 0.5% Trehalose 3%Glycine — 4% Glycine 50 mM NaCl, 0.5% Trehalose 4% Glycine 150 mM NaCl,0.5% Trehalose 4% Glycine 50 mM NaCl, 0.5% Sucrose 10 mM Sodium — 150 mMNaCl Phosphate pH 2% Glycine — 7.5 2% Glycine 0.5% Sucrose 2% Glycine0.5% Trehalose 3% Glycine — 4% Mannitol 50 mM NaCl, 0.5% Trehalose 4%Mannitol 150 mM NaCl, 0.5% Trehalose 4% Mannitol 50 mM NaCl, 0.5%Sucrose

This initial lyophilization feasibility study demonstrated that thetarget construct was more stable at pH 7.5 with sucrose and glycine withrespect to reduced aggregation. Based on the results from this initialstudy a second lyophilization screen was conducted using theformulations listed in TABLE 10.

TABLE 10 Summary of secondary formulation buffer screen BufferingPre-Lyophilization (%) Post-Lyophilization (%) Buffer Agent StabilizerSurfactant pH HMW Dimer Monomer HMW Dimer Monomer AMT-10 (StartingMaterial) 2.6 89.2 8.2 10 mM 2% Glycine 0.5% 0.3% 7.5 2.9 89.8 7.4 4.389.3 6.4 Sodium Sucrose Poloxamer Phosphate 1% Sucrose 2.96 90.1 6.9 4.189 6.9 1% 2.9 90.3 6.8 3.6 89.8 6.6 Trehalose 4% Mannitol Sucrose, 3.190.1 6.8 3.9 89.5 6.6 1.3% arginine 2.6% 4.9 86.7 8.4 4.1 89.5 6.4arginine 10 mM 2% Glycine 1% Sucrose 3.15 90.3 6.6 3.7 89.8 6.4Potassium 1% 3.2 90.1 6.7 3.9 89.5 6.6 Trehalose 10 mM 1% Sucrose 7 2.890.9 6.3 3.5 89.9 6.6 Histidine 1% 2.95 90.5 6.6 3.3 90.2 6.5 Trehalose

From this screen, the two formulations that demonstrated the beststability were:

1) 10 mM potassium phosphate, 2% glycine, 1% sucrose or 1% trehalose,0.3% poloxamer 188 at pH 7.52) 10 mM histidine, 2% glycine, 1% sucrose or 1% trehalose, 0.3%poloxamer at pH 7.0.

Using these two formulations, in order to ensure that the UF/DF stepwould function as intended and that freezing at the beginning of thelyophilization process would be acceptable, a final liquid formulationfreeze/thaw screen and short-term stability study was conducted athigher protein concentrations as outlined in TABLE 11 below.

TABLE 11 Formulations for freeze/thaw screening Formulation 10 mMPotassium Phosphate, 2% Glycine, 1% Sucrose, 0.3% Poloxamer 188, pH 7.510 mM Potassium Phosphate, 2% Glycine, 1% Trehalose, 0.3% Poloxamer 188,pH 7.5 10 mM Histidine, 2% Glycine, 1% Sucrose, 0.3% Poloxamer 188, pH7.0 10 mM Histidine, 2% Glycine, 1% Trehalose, 0.3% Poloxamer 188, pH7.0

The target construct at a concentration of 20 mg/mL did not show anysignificant increases in aggregation during freeze/thaw or at 1 week at4° C. for any condition tested. The formulation buffer consisting of 10mM potassium phosphate, 2% glycine, 1% sucrose, 0.3% poloxamer 188 at apH of 7.5 was determined to have the best overall stability at 2-8° C.and 25° C. over one week (see data in FIG. 24C and FIG. 24D, box andarrows highlighting the best formulation). This formulation wasrecommended to proceed as the buffer to be used in the UF/DF formulationstep prior to lyophilization. Data from freeze/thaw experiments areshown in FIG. 23A and FIG. 23B. Data from a short-term stability studyare shown in FIGS. 24A-24D.

Bulk lyophilization was carried out by thawing and dispensing the liquidintermediate into trays that were loaded into a lyophilizer. Controlparameters during the lyophilization cycle such as temperature andvacuum pressure were executed based on time. In-process samples weretaken after completion of the cycle. The lyophilized powder was pooledand mixed in a low-density polyethylene (LDPE) primary liner that wasplaced inside a secondary LDPE liner. The second liner was heat sealed,then placed into a mylar bag, which was also heat sealed. Whenlyophilized, the target construct resulted in a white to off-whitepowder. The combined lyophilized powder is the target construct drugsubstance (DS).

Example 6: In Vitro Evaluation of Coating Formulations

Different formulation types can be used to facilitate a targeteddelivery of an active pharmaceutical ingredient (API) on its desiredsite of action and to protect the API against certain physiologicalconditions that are present in the gastrointestinal tract which couldimpact its stability. Upon ingestion, the API comes into contact withthe low gastric pH and the proteolytic pepsin in the stomach which couldinfluence its stability. Following passage through the stomach, the APIenters the small intestine which is characterized by higher pH values.The secretion of bile salts and the proteolytic pancreatic enzymes canhave a huge impact on the stability of the API. Furthermore, the gastricpH and concentrations of proteolytic enzymes vary considerably betweenthe fed and fasted state. As such, the aim of this example was toinvestigate the disintegration of five different formulations and theirsubsequent targeted release of caffeine during passage through thecomplete gastrointestinal tract.

The reactor setup used in this experiment was adapted from the Simulatorof the Human Intestinal Microbial Ecosystem (SHIME®), representing thegastrointestinal (GI) tract of the adult human, as described by Molly etal. (Appl Microbiol Biotechnol 39:254-258(1993)), which is hereinincorporated by reference. In this system, the first two reactorssimulated different steps in food uptake and digestion, with peristalticpumps adding a defined amount of feed and pancreatic and bile liquid,respectively to the stomach and small intestine compartment and emptyingthe respective reactors after specified intervals. The last threecompartments, continuously stirred reactors with constant volume and pHcontrol, simulated the ascending, transverse, and descending colon.Retention time and pH of the different vessels are chosen in order toresemble in vivo conditions in the different parts of the GI tract.

In this experiment an adapted SHIME® system representing thephysiological conditions of the stomach and small intestine within thesame reactor over time was used (FIG. 3). In order to mimic fed orfasted conditions, a gastric suspension was added to the reactor. Afterthis, a standardized enzyme and bile liquid was added to simulate thesmall intestinal condition. Incubation conditions (pH profiles,incubation times) were optimized in order to resemble in vivo conditionsin the different regions of the gastrointestinal tract for fasted or fedconditions.

Protocol for Simulation of the Stomach and Small Intestines

During the study, the dissolution of capsules was tested during passagethrough the stomach and small intestines under fasted conditions.

In the gastric phase, the incubation occurred during 45 minutes at 37°C., while mixing via stirring, at pH 2.0 (FIG. 3). 4-fold lower pepsinand phosphatidylcholine levels were added relative to the fedconditions. As the background medium, only salts and mucins weresupplied. Sampling and visual scoring at t=0 and 45 minutes of stomachincubation.

In the small intestinal phase, while mixing via stirring, the pHinitially automatically increases from 2.0 to 5.5 within a period of 5minutes after which the pH of the medium increased from 5.5 till 6.5during the first hour, from 6.5 till 7.0 during the second hour, andremained constant at a value of 7.0 during the third hour of smallintestinal incubation (FIG. 5). The temperature was controlled at 37° C.Regarding pancreatic enzymes, both a raw animal pancreatic extract(pancreatin) containing all the relevant enzymes in a specific ratio aswell as defined ratios of the different enzymes was used. Under fastedconditions, 5-fold lower levels of the pancreatic enzymes were added ascompared to experiments performed under fed conditions. Regarding bilesalts, 3.3 mM bovine bile extract was generally supplemented as bovinebile is a closer match to human than porcine in terms of tauro- andglycocholate.

Protocol for Simulation of Colon

During this study, a fecal sample of one donor was harvested and storedat −80° C. until further use, as a source of colonic microbiotic for useduring all colonic incubations that followed the passage through theupper GI tract. The use of the same colonic microbiotic hence allowedcomparison of results obtained during the different experiments. Afterdonation of the fecal sample in a sampling box, an Anaerogen bag wasadded and the box was immediately sealed. The powder in the Anaerogenbag immediately removed all oxygen from the sampling box. Subsequently,anaerobic PBS was added to the fecal sample and a fecal slurry wasprepared by homogenization in a stomacher. The fecal slurry was brieflycentrifuged to remove large particles. Afterwards, an equal volume ofcryoprotectant solution was added to the fecal supernatant. Afterhomogenization, the cryoprotected fecal slurry was snap-frozen in liquidnitrogen and stored at −80° C.

Before starting the actual colonic experiments, the cryopreserved fecalsample was pre-incubated in bioreactors in order to obtain a fullymetabolically active colonic background microbiota that was used toinoculate the colonic incubations. Briefly, 2.5% (vol/vol) of fecalslurry was inoculated in a rich colonic medium containing both host- anddiet-derived substrates. The vessels were made anaerobic throughflushing with nitrogen gas and were incubated for 24 h at 37° C. Assuch, a fully established and metabolically active colonic microbiotawas obtained after 24 h of incubation.

After taking the SIend (small intestine end or ileum) at the end of thestomach/small intestine experiments, the colonic incubations wereinitiated. This was done by adding 200 mL of fresh colonic medium,containing host- and diet-derived substrates, to the 200 mL ofstomach/small intestine suspension. The simulation of a metabolicallyactive luminal colonic microbiota was obtained by adding 300 mL of thepre-incubated fecal material to the bioreactors. The vessels were madeanaerobic by flushing with nitrogen gas and were subsequently incubatedfor 18 h at 37° C. Visual scoring of the capsules and sampling of thereactors was performed after 0; 0.5; 1; 1.5; 2; 3; 4 h; and 18 h ofcolonic incubation.

Disintegration of Capsules

During transit in the simulated GI tract, a visual inspection of thecapsules was conducted according to the following score: 1: capsuleintact; 2: capsule damaged but almost all product is still in thecapsule; 3: capsule damaged and all product was released; 4: capsuledestroyed.

An HPLC-UV/Vis method was implemented that allowed to quantify theconcentration of caffeine in the samples taken from the reactors.Briefly, the samples were run using an isocratic separation method (25%methanol: 75% water) on a C18 column. The column temperature wascontrolled at 25° C. The total run time per sample was 7 min. Theinjection volume was 10 μL and the UV/Vis detector was operated at 272nm. Quantification of caffeine was performed using external standards.Prior to injection on the column, the samples were centrifuged for 15min at 9000 rpm. Subsequently, the supernatant was filtered through a0.2 μm filter into HPLC vials.

Statistically significant differences between the concentration ofcaffeine was determined in between each sampling point and its precedingone during the experiments under fasted conditions to demonstratechanges in function of time. In terms of statistics, the differences forall data discussed and indicated by “p<0.05” or “*” were significantwith a confidence interval of 95%, as demonstrated using a Student'st-test.

The disintegration of five different formulation types during passagethrough the GI tract was investigated (TABLE 12). Next to theexperiments with the five formulations a control experiment wasperformed to determine the concentration of caffeine in the background.To each reactor, one capsule was added and the capsules were mounted ina capsule sinker. All experiments were performed in biologicaltriplicate.

TABLE 12 Formulations tested Eudragit ® L30D55: FIG. showing CapsuleEudragit ® Capsule Coating caffeine release Identity FS30D ratioContents Thickness profile A 50:50 20 mg SEQ ID  60 mg FIG. 5A NO: 5 10mg caffeine B 50:50 20 mg SEQ ID 128 mg FIG. 5B NO: 5 10 mg caffeine C20:80 10 mg rHSA  60 mg FIG. 5C 10 mg caffeine D 20:80 10 mg rHSA 120 mgFIG. 5D 10 mg caffeine E  0:100 10 mg rHSA  60 mg FIG. 5E 10 mg caffeine

Determination of the concentration of caffeine present at the differentsampling points during the control experiments revealed that thiscompound was not present in the stomach, small intestinal and colonicphase of the GI tract passage experiments. Hence, the background mediaused during the experiments with the five formulations could notgenerate interference with the detection of caffeine released from thecapsules.

Capsule 1, having a coating thickness of 60 mg on a size 1 capsule and aEudragit® ratio L30D55:FS30D of 50:50, remained completely intact duringpassage through the simulated fasted stomach, thereby protecting the APIagainst the low pH conditions present during the stomach incubationphase. The capsules remained visually intact during the first hour ofsmall intestinal incubation during which the pH of the medium increasedfrom a value of 5.5 till 6.5. The capsules became damaged during thesecond hour of small intestinal incubation during which the pH increasesfrom a value of 6.5 till a value of 7.0. FIG. 5A illustrates release ofcaffeine from Capsule 1.

Notwithstanding the visual damage to the capsule, the majority of thepowder remained in the capsules as was demonstrated by the low amountsof caffeine measured after 2 hours of small intestinal incubation. Thecapsules became even more damaged during the third hour of smallintestinal incubation (stable pH of 7.0) resulting in the release of amajor part of the powder inside the capsules as was evident by thequantification of caffeine at this sampling point. During the colonicincubations the amount of caffeine remained fairly constant. A smallincrease was observed and this mainly due to the incomplete release ofcaffeine from the capsule during the experiments of replicate 2. Thecapsules were not completely destroyed at the end of the colonicincubation phase and visual inspection of the capsules revealed that aminor part of the powder was still present inside the capsules. Thisexplains the reason why the total dose of 10 mg caffeine, which waspresent in the capsules, was almost fully released by the end of thecolonic incubation. As such, it can be concluded that capsule 1facilitates a targeted delivery of an API at the end-stages of the smallintestinal incubation phase which corresponds with the terminal ileum ofthe gastrointestinal tract (GIT).

Capsule 2, having a coating thickness 128 mg on a size 1 capsule and anEudragit® L30D55:FS30D ratio of 50:50, remained completely intact duringpassage through the fasted stomach (FIG. 5B).

Furthermore, as compared to capsule 1 (same Eudragit® ratio) theincreased thickness of the coating of capsule 2 prevented the capsule tobecome damaged during passage through the small intestinal incubationphase where the pH of the medium increases from a value from 5.5 till7.0.

Entrance of the capsule into the colonic environment resulted in visualdamage to the capsules after 1.5 hours of incubation which resulted in asmall release of caffeine after 4 hours of colonic incubation. Prolongedcolonic incubation induced further damage to the capsules resulting inthe release of high amounts of caffeine into the colonic lumen after 18hours of colonic incubation. Throughout the colonic incubation phase thepH of the medium was controlled above a value of 5.8. As such, it can beconcluded that the increased thickness of the coating of capsule 2resulted in a delayed release of caffeine as compared to capsule 1. Thecapsules were not completely destroyed after the passage through the GItract indicating that one of the polymers of the capsules did notdissolve at the pH values that were present during the GI tract passage.As such, it can be concluded that capsule 2 facilitates a targeteddelivery of an API at the end-stages of the colonic incubation phasewhich corresponds with the distal colon of the GI tract.

Capsule 3, having a coating thickness of 60 mg on a size 1 capsule andan Eudragit® L30D55:FS30D of 20:80, remained completely intact duringpassage through the fasted stomach (FIG. 5C). Whereas the capsules didnot become visually damaged during passage through the small intestine,small amounts of caffeine were detected after 3 hours of smallintestinal incubation indicating the occurrence of undetectablemicroscopic damage to the capsules. The capsules became visually damagedafter 0.5 hours of colonic incubation which resulted in increasedamounts of caffeine being released from the capsules after 4 hours ofcolonic incubation. The amount of caffeine detected in the colonicmedium further increased in between 4 h and 18 h of colonic incubation.The capsules did not become fully destroyed at the end of the passagethrough the full GI tract. As such, comparison of the data obtainedduring the experiments with capsule 1 and 3 indicated that increasingthe percentage of FS30D at the cost of L30D55 resulted in the targeteddelivery of the API at the start of the colonic incubation phase whichcorresponds to the proximal colon of the GI tract.

Capsule 4, having a coating thickness of 120 mg on a size 1 capsule anda Eudragit® L30D55:FS30D ratio of 20:80, remained visually intactthroughout the passage of the complete GI tract (FIG. 5D). Only duringthe experiments of replicate 2 and 3, a minor amount of caffeine wasdetected. Hence, increasing the coating thickness and ratio of FS30D atthe cost of L30D55 prevented the release of the API in the upper GItract and proximal colon. It could be hypothesized that the contentwould only be released towards the distal colon upon longer incubationtimes at increasing pH.

Capsule 5, having a coating thickness of 60 mg on a size 1 capsule and aEudragit® L30D55:FS30D ratio of 0:100, became visually damaged after 3hours of small intestinal incubation when the pH of the small intestinalsuspension was equal to 7 (FIG. 5E). However, nearly all powder remainedtrapped inside the capsule as was demonstrated by the absence ofmeasurable amounts of caffeine at the end of the small intestinalincubation phase. Upon entering the colon, the capsules did not becomefurther visually damaged. Only during the replicate 3 experimental runcaffeine was detected at an adequate amount after 18 hours of colonicincubation. As such, it can be concluded that omitting L30D55 out of thecapsule polymer mixture resulted in the absence of a targeted deliveryof the API during passage through the upper GIT and proximal colon. Itcould be hypothesized that the content would only be released towardsthe distal colon upon longer incubation times at increasing pH.

Conclusion

During the present study, the disintegration of five differentformulations during passage through the stomach, small intestine, andcolon was evaluated. Dissolution of the capsules was studied throughvisual scoring at dedicated time points and through determination of theamount of caffeine released from the capsules during passage through thegastrointestinal tract (GIT).

All five formulations displayed different dissolution characteristicswhich were determined by their coating thickness and Eudragit®L30D55:FS30D ratio. The capsules of the present study with a coatingthickness of 60 mg facilitated a targeted delivery of the API at the endof the small intestinal incubation or the beginning of the colonicincubation when their Eudragit® L30D55:FS30D ratio was 50:50 or 20:80,respectively. Increasing the coating thickness of capsules with anEudragit® L30D55:FS30D ratio of 50:50 from 60 mg till 128 mg resulted inthe targeted delivery of the API at the end stages of the proximalcolonic incubations. Whereas capsules 1, 2, 3 were capable to provide atargeted delivery of the API, these capsules did not become completelydissolved at the end of the passage through the GIT resulting in thepresence of capsule material at the end stages of the colonicincubations. Capsules 4 (coating thickness 120, Eudragit® L30D55:FS30Dratio of 20:80) and capsule 5 (coating thickness of 60 mg and Eudragit®L30D55:FS30D ratio of 0:100) did not disintegrate during passage throughthe upper GIT and proximal colon resulting in the absence of thetargeted delivery of the API during the current experiment. It could behypothesized that the content would only be released towards the distalcolon upon longer incubation times at increasing pH.

Example 7: Evaluation of Enteric Coating

The composition of the enteric coat was selected based on experimentsconducted on research batches of enterically coated capsules. Thecomposition of these research batches is summarized in TABLE 13. Size 1HPMC capsules were used for all batches. The fill weight of rHSA(recombinant human serum albumin) or the substance powder wasapproximately 30 mg as the protein content of each was approximately onethird of the powder weight. Coating compositions comprised mixtures ofEudragit® L30D55 with a nominal dissolution pH of >5.5, and Eudragit®FS30D with a nominal dissolution pH of >7 (Evonik GmbH productinformation). Capsule release at a pH value of approximately 6.5 wasdesired to provide adequate enteric protection while allowing release ofthe target construct in the intestine.

TABLE 13 Composition of capsules for formulation development. Allbatches were filled to size 1 HPMC capsules. Eudragit coating Entericcoating composition (ratio of target weight L30D55:FS30D) (mg) Capsulecontents 50:50 60 Caffeine (10 mg), rHSA (10 mg) 30:70 120 Caffeine (10mg), IL-10 delivery 20:80 180 construct (SEQ ID NO: 5)  0:100 (10 mg)

Coating evaluation was conducted in two research studies with multiplebatches of coated capsules.

For the first study, twelve batches of capsules containing caffeine andrecombinant human serum albumin (rHSA) were prepared, comprisingpermutations of four different coating compositions and three differentcoating thicknesses. The coating compositions comprised ratios ofEudragit® L30D55 and Eudragit® FS30D, between 50:50 to 0:100 by weight.

Caffeine was included in this study as an easily-detected marker forcapsule release. rHSA was considered to be a suitable surrogate proteinfor the target construct in this study as it was prepared as alyophilized composition using the same lyophilization buffer as used forthe target construct drug substance, at approximately the same proteincontent as the target construct drug substance, and the physical form ofthe lyophilized composition is comparable.

For the second study, three batches of capsules containing caffeine andthe target construct were prepared, comprising three different coatingthicknesses of a coating formulation containing equal amounts ofEudragit® L30D55 and Eudragit® FS30D.

Capsules from each research batch were placed in a stirred solution of0.1 N HCl for at least 60 min, followed by transfer to buffer solutionsat specified, higher pH values. These conditions were intended tosimulate exposure to the acid environment of the stomach, followed byapproximately neutral pH on passage to the intestines. In eachexperiment, the supernatant was periodically sampled and tested for theconcentration of capsule contents which have been released from thecapsule into solution.

Results of these experiments are summarized below. In no instance didany enterically coated capsule release contents during the 1 hincubation phase in 0.1 N HCl. Thus, data presented in the tablesrepresents release during the buffer incubation phase only. As expected,enteric protection from acidic environment is demonstrated by allcoatings evaluated.

First Study: Selection of Coat Composition

The composition of the enteric coat was selected from an initial studyusing capsules containing caffeine as a release marker and rHSA as aprotein surrogate for the target construct. The ratio of Eudragit®L30D55 and Eudragit® FS30D varied between 50:50 to 0:100 to explore theeffect of coating composition on capsule release as a function ofsolution pH.

TABLES 14-17 show the behavior of 12 sets of coated capsules containingcaffeine and rHSA in pH 7.0 buffer. Caffeine and rHSA values werenormalized to 100% for capsules reaching maximum release, otherwise datawas unadjusted. The release kinetics of caffeine and rHSA varied basedon the weight and composition of capsule coating, although the releaseof both compounds was comparable for each individual capsule batch. Thuscaffeine (a small molecule) and rHSA (a protein) provided similarinformation about capsule release under these conditions.

TABLE 14 Percent release of caffeine and rHSA from capsules coated with50:50 ratio of Eudragit ® L30D55 and Eudragit ® FS30D, pH 7.0 BufferCaffeine release at stated time rHSA release at stated time Coatingweight (mg) 1 h 2 h 3 h 4 h 6 h 8 h 1 h 2 h 3 h 4 h 6 h 8 h 60 6 84 8787 88 90 41 97 97 100 97 98 125 2 6 77 95 98 100 0 5 94 97 98 100 180 12 3 56 97 100 0 0 4 53 100 100

TABLE 15 Percent release of caffeine and rHSA from capsules coated with30:70 ratio of Eudragit ® L30D55 and Eudragit ® FS30D, pH 7.0 BufferCoating Caffeine release at stated time rHSA release at stated timeweight mg 1 h 2 h 3 h 4 h 6 h 8 h 1 h 2 h 3 h 4 h 6 h 8 h 61 15 95 96 9899 100 24 98 98 98 98 100 118 1 4 82 91 95 100 0 3 92 95 98 100 182 0 13 55 93 100 0 0 0 74 94 100

TABLE 16 Percent release of caffeine and rHSA from capsules coated with20:80 ratio of Eudragit ® L30D55 and Eudragit ® FS30D, pH 7.0 BufferCaffeine release at stated time rHSA release at stated time Coatingweight (mg) 1 h 2 h 3 h 4 h 6 h 8 h 1 h 2 h 3 h 4 h 6 h 8 h 56 5 87 9797 100 98 7 96 98 97 99 100 120 1 2 7 78 95 100 0 0 2 89 98 100 177 1 11 5 56 100 0 0 0 2 87 100

TABLE 17 Percent release of caffeine and rHSA from capsules coated with0:100 ratio of Eudragit ® L30D55 and Eudragit ® FS30D, pH 7.0 BufferCaffeine release at stated time rHSA release at stated time Coatingweight (mg) 1 h 2 h 3 h 4 h 6 h 8 h 1 h 2 h 3 h 4 h 6 h 8 h 59 1 19 9397 99 100 0 50 97 98 98 100 120 1 1 3 23 83 100 0 0 0 0 94 100 181 1 2 2ND 2 18 0 0 0 0 0 45

For these capsule batches, greater total weight of enteric coatcorrelated with a slower onset of release of caffeine and rHSA in pH 7.0buffer, and longer time to achieve complete release. Less significantcorrelation was seen between release in pH 7.0 buffer and the coatingcomposition. Almost all capsules released their contents completely inpH 7.0 buffer over the course of testing, with the exception thatcapsules coated with 100:0 ratio of FS30D to L30D55 showed delayed onsetof release and incomplete release for greater coating weights.

TABLES 18-19 summarize the behavior of the same capsules in pH 6.5 andpH 6.0 buffers. Only rHSA values are presented, as the kinetics ofcaffeine release and rHSA were again comparable for each capsule. rHSAvalues were normalized to 100% for capsules reaching maximum release,otherwise data was unadjusted. Coat weight shown in TABLES 18-19 was thetarget coating weight, but actual coating weight varied by not more than5 mg from the target coating weight.

TABLE 18 Release of rHSA from capsules coated with different ratios ofEudragit ® L30D55 and Eudragit ® FS30D, pH 6.5 Buffer Release of rHSAfrom Capsules of Stated Coating Composition at Stated Time CoatingComposition 50:50 FS30D/L30D55 70:50 FS30D/L30D55 80:50 FS30D/L30D55Coating weight (mg) 1 h 2 h 3 h 4 h 6 h 8 h 1 h 2 h 3 h 4 h 6 h 8 h 1 h2 h 3 h 4 h 6 h 8 h 60 11 93 96 98 99 100 8 78 95 97 98 100 0 17 64 7782 92 120 0 2 13 94 98 100 0 7 36 64 92 100 0 0 0 0 12 42 180 0 0 2 3 94100 0 0 0 4 43 85 0 0 0 0 1 10

TABLE 19 Release of rHSA from capsules coated with different ratios ofEudragit ® L30D55 and Eudragit ® FS30D, pH 6.0 Buffer Release of rHSAfrom Capsules of Stated Coating Composition at Stated Time CoatingComposition 50:50 FS30D/L30D55 70:30 FS30D/L30D55 80:20 FS30D/L30D55Coating weight (mg) 1 h 2 h 3 h 4 h 6 h 8 h 1 h 2 h 3 h 4 h 6 h 8 h 1 h2 h 3 h 4 h 6 h 8 h 60 12 79 97 98 100 100 4 30 43 58 62 67 0 0 29 39 52100 120 0 6 21 23 83 98 0 0 0 0 39 72 0 0 0 0 0 0 180 0 0 0 0 6 30 0 0 00 0 3 0 0 0 0 0 0

In either buffer condition, a clear trend in release kinetics withrespect to coating weight was observed whereby increased total weight ofenteric coat correlated with a slower onset of release of rHSA, andlonger time to achieve complete release. At pH 6.5 and pH 6.0, a trendin coating composition was also evident. Coatings containing a higherproportion of Eudragit® FS30D showed delayed onset of release with lowerbuffer pH, and incomplete or no release was observed for greater coatingweights in these cases. No release was observed for any capsule coatedwith 100:0 ratio of FS30D to L30D55 under these conditions at pH 6.5 or6.0.

Thus, the release of rHSA was dependent on both the weight andcomposition of the enteric coat. A coating weight of 60 mg with a 50:50composition of Eudragit® L30D55 and Eudragit® FS30D provided the mostrapid release of capsules tested under these conditions.

Second Study: Selection of Coat Weight

Evaluation of the coating weight was continued in a second study withcapsules containing target constructs.

TABLES 20-22 show the behavior of three batches of capsules containingcaffeine and target constructs, with different coating weight of 50:50Eudragit® polymers L30D55 and FS30D. The release of caffeine and targetconstructs in buffers at pH 7.0, pH 6.5, and pH 6.0 was examined. Thesecapsules were subjected to prior incubation in 0.1 N HCl for 1 h, and norelease of caffeine or target construct was detected in any instance.Caffeine and target construct values were normalized to 100% forcapsules reaching maximum release, otherwise data was unadjusted. NDindicates data point could not be determined due to sample loss.

TABLE 20 Release of caffeine and target constructs from capsules coatedwith 50:50 ratio of Eudragit ® L30D55 and Eudragit ® FS30D, pH 7.0Buffer Caffeine release at stated time AMT-101 release at stated timeCoating weight (mg) 1 h 2 h 3 h 4 h 6 h 8 h 1 h 2 h 3 h 4 h 6 h 8 h 6013 100 97 97 98 100 8 81 81 81 78 75 128 0 3 60 95 98 100 0 0 60 81 81ND 178 0 1 3 5 76 100 0 0 6 6 45 39

TABLE 21 Release of caffeine and target constructs from capsules coatedwith 50:50 ratio of Eudragit ® L30D55 and Eudragit ® FS30D, pH 6.5Buffer Caffeine release at stated time AMT-101 release at stated timeCoating weight (mg) 1 h 2 h 3 h 4 h 6 h 8 h 1 h 2 h 3 h 4 h 6 h 8 h 60 522 97 98 98 100 0 60 72 72 73 70 128 0 1 6 36 81 100 0 0 0 32 41 39 1780 0 0 2 11 37 0 0 0 0 0 5

TABLE 22 Release of caffeine and target constructs from capsules coatedwith 50:50 ratio of Eudragit ® L30D55 and Eudragit ® FS30D, pH 6.0Buffer Caffeine release at stated time AMT-101 release at stated timeCoating weight (mg) 1 h 2 h 3 h 4 h 6 h 8 h 1 h 2 h 3 h 4 h 6 h 8 h 60 316 56 88 98 100 7 13 18 18 17 17 128 0 0 3 8 51 88 0 0 0 0 6 6 178 0 0 02 11 34 0 0 0 0 0 0

The time to onset of release of caffeine and target constructs werecomparable for each capsule. As seen earlier for the rHSA capsules, aclear trend in release kinetics with respect to coating weight wasobserved whereby increased total weight of the enteric coat correlateswith a later onset of release of caffeine and target constructs, andlonger time to achieve complete release. In general, release of targetconstructs reached concentrations which were lower than calculated basedon the capsule fill weight. Higher concentrations of target constructswere generally achieved for capsules with an earlier time of onset ofrelease. The reason for lower than expected release of target constructswill be investigated in future studies. At this early stage indevelopment, fast-releasing coating compositions were selected tominimize potential loss of target constructs during capsule release.

Selection of Enteric Coat

Based on the research capsule studies, a coating composition comprising50:50 mixture of Eudragit® polymers L30D55 and FS30D was selected forthe clinical capsule presentation. A target coating weight of 60 mg onSize 1 capsule was selected to provide enteric protection from stomachacid, but release capsule contents on reaching a neutral pH environment.Coating weight of 60 mg on a Size 1 capsule provides equivalent coatthickness to coating of 75 mg for a Size 0 capsule selected for clinicalpresentation.

Example 8: In Vitro Dissolution Data

Eight capsule coating formulations (Formulations A-H in TABLE 23) weretested for in vitro dissolution rates at varying pHs. The first hour wasan acid stage where the capsule was exposed to a dissolution mediacontaining 0.1 M hydrochloric acid at a pH of 1.0. The remaining hourswere spent in a buffer stage where the capsule was exposed to adissolution media containing a citrate/phosphate buffer at pH of 7.0,6.5, or 6.0. Each capsule was removed using a plastic spatula whilechanging the media. Capsules were placed inside a 150 mL glass beakerwith a stir bar stirring at 100 rpm and a heater set up at 37° C. Thepercent release of caffeine was determined by measuring UV absorbance.The percent release of the IL-10 delivery construct was determined bysize exclusion chromatography (SEC) (TABLE 24) and the dimer form wasdetected as a single peak. Recorded values were determined from astandard curve of the respective analyte. During SE-HPLC, multi-anglelight scattering (MALS) detection in combination with UV absorbance andrefractive index (RI) detection is used to determine the molecular massof the eluted peaks. Detection was performed by absorbance at 280 nm.

Eudgragit-coated capsules were subject to 1 hour of acid stagedissolution and then buffer stage dissolution. Capsule without aEudragit coat (HPMC coat only) were test in the buffer stage only.Dissolution media was stirred at 100 rpm for the entire duration of theassay. 500 μL aliquots of samples were collected at the end of the acidstage, 1 h, 2 h, 3 h, 4 h, 6 h, and 24 h of the buffer stage into 0.22μm cellulose acetate Spin-X centrifuge tube filters (Costar Cat #8161).Centrifugation was done at 15,000×g for 2 min. 150 μL aliquots ofsamples were then transferred into HPLC vials and analyzed by first SECand then RP chromatography.

TABLE 23 Formulation of capsules examined for in vitro dissolution ofcapsule contents Target construct Caffeine Eudragit ® amount amount HPMCEudragit ® Coat Formulation (mg) per (mg) per Capsule Composition WeightIdentifier capsule capsule Coat (mg) (L30D55/FS30D) gain Description A10 10 10 50/50 60 Reference composition B 10 10 10 50/50 30 ThinEudragit ® 50/50 C 10 10 10 30/70 30 Thin Eudragit ® 30/70 D 10 10 1030/70 60 Standard 30/70 E 10 10 10 30/70 90 Standard 30/70 F 10 10 1030/70 120 Thick 30/70 G 10 10 60 50/50 30 Increase HPMC H 10 10 18050/50 30 Thick HPMC

TABLE 24 SE-HPLC Method System Vanquish UHPLC system with PDA DetectorColumn Waters ACQUITY UPLC Protein BEH SEC 200Å 1.7 μm, 4.6 × 150 mmPart No: 186005225 Column Temperature 25° C. Autosampler Temperature  4°C. Mobile Phase 100 mM Sodium Phosphate, 150 mM Sodium Chloride, pH 7.0± 0.1 Separation mode Isocratic Flow Rate 0.3 mL/min Total Runtime 10min Detection Wavelengths 215 nm, 280 nm Injection Volume 100 μL (orvary)

Percent of caffeine released at pH 7.0 was measured for each of capsuleformulations A-B (FIG. 6A), capsule formulations C-F (FIG. 6B), andcapsule formulations G-H (FIG. 6C). Percent of caffeine released at pH6.5 was measured for each of capsule formulations A-B (FIG. 7A), capsuleformulations C-F (FIG. 7B), and capsule formulations G-H (FIG. 7C).Percent of caffeine released at pH 6.0 was measured for each of capsuleformulations A-B (FIG. 8A), capsule formulations C-F (FIG. 8B), andcapsule formulations G-H (FIG. 8C).

Percent of target construct released at pH 7.0 was measured for each ofcapsule formulations A-B (FIG. 9A), capsule formulations C-F (FIG. 9B),and capsule formulations G-H (FIG. 9C). Percent of target constructsreleased at pH 6.5 was measured for each of capsule formulations A-B(FIG. 10A), capsule formulations C-F (FIG. 10B), and capsuleformulations G-H (FIG. 10C). Percent of target constructs released at pH6.0 was measured for each of capsule formulations A-B (FIG. 11A),capsule formulations C-F (FIG. 11B), and capsule formulations G-H (FIG.11C).

The percent of the released target constructs in the dimer form was alsodetermined. Percent of released target construct in the dimer form at pH7.0 was measured for each of capsule formulations A-B (FIG. 12A),capsule formulations C-F (FIG. 12B), and capsule formulations G-H (FIG.12C). Percent of released target constructs in the dimer form at pH 6.5was measured for each of capsule formulations A-B (FIG. 13A), capsuleformulations C-F (FIG. 13B), and capsule formulations G-H (FIG. 13C).Percent of released target constructs in the dimer form at pH 6.0 wasmeasured for each of capsule formulations A-B (FIG. 14A), capsuleformulations C-F (FIG. 14B), and capsule formulations G-H (FIG. 14C).

Example 9: Capsule Coating Study in Cynomolgus Monkeys

Eight capsule coating formulations (Formulations A-H in TABLE 25) weretested in male cynomolgus monkeys. Capsules were administered as asingle dose, with 2 capsules per animal, and were orally administeredwith a pill gun. Plasma samples were collected 8 hours post capsuleadministration and analyzed for IL-10 (FIGS. 15A, 16A, and 17A),caffeine (FIGS. 15B, 16B, and 17B), and interleukin-1 receptorantagonist (IL-IRA) (FIGS. 15C, 16C, and 17C).

TABLE 25 Formulation of capsules administered to cynomolgus monkeysTarget Caffeine Number construct amount HPMC Eudragit ® of amount (mg)Capsule Eudragit ® Coat capsules Formulation (mg) per per CoatComposition Weight per Identifier capsule capsule (mg) (L30D55/FS30D)gain Description animal N A 10 10 10 50/50 62 Reference 2 3 compositionB 10 10 10 50/50 31 Thin 2 3 Eudragit ® 50/50 C 10 10 10 30/70 31 Thin 23 Eudragit ® 30/70 D 10 10 10 30/70 62 Standard 30/70 2 3 E 10 10 1030/70 92 Standard 30/70 2 3 F 10 10 10 30/70 123  Thick 30/70 2 3 G 1010 60 50/50 31 Increase 2 3 HPMC H 10 10 180  50/50 31 Thick HPMC 2 3

A robust caffeine signal indicated capsule opening behavior. Capsuleopening time and kinetics (from caffeine) correlated well with in vitrodissolution data. Thinner coats showed the most rapid opening (30 mgcoat of 50/50 Eudragit® L30D55/FS30D and 30/70 Eudragit® L30D55/FS30D).Systemic 11-10 and IL-IRA levels were elevated for some formulations.Time course of PK and biomarker signals correlated well with caffeinerelease time course data. Thinner coats showed the most significantelevation of systemic IL-10 and IL-1RA (30 mg coat of 50/50 Eudragit®L30D55/FS30D and 30/70 Eudragit® L30D55/FS30D).

Example 10: Development of Powder with Improved Characteristics

Lyophilized composition compositions of low density can have poor flowcharacteristics. The goal was to develop a lyophilized compositionformulation with increased density and improved flow properties. Thelyophilized composition was blended with excipients to improve capsulefilling or enable tablet formulation.

Recombinant human serum albumin (rHSA) was used as a surrogate proteinfor filing. Lyophilized rHSA (lyo-rHSA) was made with the same process,composition, and density as lyophilized target constructs, with a targetof 20 mg API per capsule (equivalent to 56 mg lyophilized composition).

The Profill capsule filling system was used to generate seven differentblends (TABLE 26), including a lyophilized drug substance (containingonly lyophilized rHSA) as well as the lyophilized drug substance inaddition with other excipients, such as glycine and sucrose.

TABLE 26 Profit blends % % Target Fill Target Filled AcceptableAcceptable Weight Capsule Capsules ± Capsules ± Mixture (mg) Weight (mg)5% 7.5% 100% lyo-rHSA 56.4 118.0 53 82 95% lyo-rHSA, 5% Glycine 59.4121.0 65 78 90% lyo-rHSA, 10% Glycine 62.6 124.4 45 63 80% lyo-rHSA, 20%Glycine 70.4 132.0 29 42 95% lyo-rHSA, 5% 2:1 Sucrose:Glycine 59.4 121.061 75 90% lyo-rHSA, 10% 2:1 Sucrose:Glycine 62.8 124.4 57 66 80%lyo-rHSA, 10% 2:1 Sucrose:Glycine 70.4 132.0 55 69

In conclusion, ProFill was feasible system to use to fill capsules witha powder, such as the lyophilized IL-10 delivery construct. It couldalso be concluded that the addition of excipients was not necessary toachieve powder fill by ProFill. Additionally, powder from capsules whichdo not meet the weight targets can be recovered and recycled.

Example 11: Compatibility Screening of Target Construct and CompactingExcipients

The compatibility of various tablet excipients with the IL-10 deliveryconstruct (SEQ ID NO: 5) was evaluated in the dry (powder) state.

The IL-10 delivery construct in the dry state (IL-10 delivery constructdrug substance, or DS, developed using the formulation buffer previouslydetermined to have the best overall stability (10 mM potassiumphosphate, 2% glycine, 1% sucrose, 0.3% poloxamer 188 at a pH of 7.5),as described in Example 5) was blended with each individual excipientthen incubated standing at 4° C. for 72 h before analysis. Thecomposition and analytical summary of each sample is described in TABLE27. IL-TO delivery construct DS and excipient (equal weight of each, 1:3ratio of API: excipient) were placed in a capped 4 mL borosilicate glassvial and blended using TURBULA® system for 2 minutes, then samples wereincubated standing at 40° C. for 72 h in a capped 4 mL borosilicateglass vial. The powder blend was then reconstituted with PBS to 0.5mg/mL solution of the IL-T delivery construct. Samples were then passedthrough 0.2 μm filter prior to SEC analysis (FIG. 18). Noincompatibility with target constructs was observed in the powder state.

TABLE 27 Composition and SEC analysis of dry-blended IL-10 deliveryconstruct/excipient powders Composition API Weight weight SEC AnalysisSample # Excipient Grade (mg) (mg) HMW Dimer Monomer 1 DS only NA  0 1210.1 81.5 7.1 2 Silicified Prosolv 36 12 11.7 82.4 5.9 microcrystallineSMCC 90 cellulose 3 Crospovidone Kollidon CL 36 12 11.7 80.3 8   4Lactose Ligamed 36 12 12.2 81.2 6.7 monohydrate 5 MCC VIVAPUR 36 12 12.781.0 6.2 6 Glyceryl COMPRITOL 36 12 12.3 80.1 7.6 behenate 7 MagnesiumLigamed 36 12 11.5 79.2 9.3 stearate 8 Croscarmellose VIVASOL 36 12 12.979.2 7.8 soldium GF 9 Starch Aldrich 36 12 12.5 80.8 6.7

For tablet manufacturing, material was subjected to compaction andcompression processes. For a protein drug with potentially sensitivetertiary and quaternary structure, these mechanical stresses requireinvestigation for the retention of protein integrity.

The compatibility of the IL-TO delivery construct was evaluated for theinitial steps of tablet formation. Blending and compaction processeswere conducted. Both roller compaction and slugging was conducted, withsubsequent milling to granules in each case. The composition of eachformulation is shown in TABLE 28. In order to explore excipientfunctionality and compatibility, different excipients were included inthe three formulations.

TABLE 28 Compositions of initial formulations forcompaction/granulation; IG = intragranular composition; EG =extragranular composition. % values are w/w and are nominal values (notcorrected for protein content) Slugging/granulation F1 Formulation F2Formulation Formulation IG EG IG EG IG EG Component Identity (%) (%)Identity (%) (%) Identity (%) (%) Active SEQ ID 8.00 — SEQ ID 8.00 — SEQID 17.31 — ingredient NO: 5 NO: 5 NO: 5 Bulking SMCC 68.00 18.35 DCP/MCC68.00 18.35 Sucrose 72.32 — agent Disintegrant Cros- 3.00 1.00 Cros-3.00 1.00 Cros- 3.24 1.94 povidone carmellose povidone sodium LubricantMg 1.00 0.25 Mg 1.00 0.25 Glyceryl 3.24 1.94 stearate stearate behenate

The results from 2 pre-blend formulations (F1 and F2) are shown in TABLE29. Roller compaction was conducted at 2 forces, and the resultingribbons were individually granulated. Materials were reconstituted inPBS at 0.65 mg/mL SEQ ID NO: 5 and filtered with 0.2 um filter(Advantec) before SEC analysis. Samples were also incubated at 40° C.for two weeks before reconstitution and analysis.

TABLE 29 IL-10 delivery construct (SEQ ID NO: 5) dimer purity duringgranulation process for formulations F1 and F2; Roller compaction at 2forces on small-scale apparatus. Granulation through 1.2 μm screen. Roomtemperature (RT) 40° C. Dimer Purity Dimer Purity Intermediate F1 F2 F1F2 Drug substance (DS) 75.9 78.6 Pre-blend 75.1 73.7 77.3 76.3 Ribbon(low force) 72.5 74.4 76.1 74.5 Ribbon (high force) 71.6 71.1 73.7 72.6Granule (low force) 74.3 74.6 74.5 72.6 Granule (high force) 74.5 73.871.9 72.4

The dimer purity data of F1 and F2 intermediates indicated littledifference as a result of these processing steps, and the values after40° C. incubation were similar to the initial samples. Nonetheless eachprocess intermediate showed somewhat reduced dimer purity compared tothe drug substance.

The same analysis was conducted for the intermediates generated in theslugging/granulation process (TABLE 30). As well as the differentcompaction method, the slugging/granulation batch differed in binder andlubricant composition. Sample dimer purity showed little to nodifference among in-process samples, and only a slight decline in dimerpurity compared to the drug substance. This suggested that thecomposition and process of the slugging/granulation samples maintainedIL-10 delivery construct integrity.

TABLE 30 IL-10 delivery construct (SEQ ID NO: 5) dimer purity duringgranulation process for slugging/granulation formulations Drug Inter-Substance Sieved Premix Final mediate (DS) DS Blend Blend Slug GranulePurity 81.2 79.5 79.4 80.4 79.3 78.9

Selected process intermediate samples were subjected to stabilityassessment following reconstitution in PBS (FIG. 95). IL-10 deliveryconstruct (SEQ ID NO: 5) solution was stable at RT over the course ofthe experiment, but showed significant degradation when stored at 37° C.Most samples showed comparable solution stability to IL-10 deliveryconstruct (SEQ ID NO: 5) DS, but both the F1 and F2 blends showed fasterdegradation comparing to samples from the slugging/granulation process.

These results suggested that components of the F1 and F2 formulationswere less compatible with SEQ ID NO: 5, or that the compacting processmay be affecting IL-10 delivery construct (SEQ ID NO: 5).

To identify if detrimental components were present in the F1/F2composition, individual excipients were evaluated for compatibility withIL-10 delivery construct (SEQ ID NO: 5) in solution. DS and a singleexcipient were suspended in PBS at 0.3 mg/mL IL-10 delivery construct(SEQ ID NO: 5). Samples were incubated at 37° C. with shaking for 5 h,with samples being periodically withdrawn for SEC analysis. The resultsare shown in FIG. 96.

Most samples showed comparable degradation to IL-10 delivery construct(SEQ ID NO: 5) DS, but the presence of magnesium stearate, used aslubricant in F1/F2 compositions, caused a significant increase in IL-10delivery construct (SEQ ID NO: 5) degradation rate. Notably, glyceryldibehenate did not show adverse effect on IL-10 delivery construct (SEQID NO: 5) dimer purity.

Magnesium stearate has ionic surfactant-like properties, whereasglyceryl behenate is a non-ionic surfactant. To further characterizelubricant compatibility a series of additional lubricant candidates wereevaluated. The results are shown in FIG. 97.

Sodium stearyl fumarate, a fatty acid salt related to magnesiumstearate, caused slightly accelerated degradation of IL-10 deliveryconstruct (SEQ ID NO: 5). Sodium laurisulfate, with the strongly anionicsulfate group, degraded IL-10 delivery construct (SEQ ID NO: 5)immediately upon mixing.

Example 12: Screening of Compression Forces for Tablet Development

Various compression forces were used to create F1 tablets (TABLE 31) andF2 tablets (TABLE 32). Dimer purity of the target construct was alsoexamined following compaction into a tablet. The target constructprotein was robust to compaction and granulation, although someaggregation was observed in final tablet form (FIG. 19). Dimer recoverywas also assessed following dissolution of various tablets at pH 7.0(FIGS. 20A-20D).

TABLE 31 F1 tablet properties Friability Compression Thickness, Screen(n = 1), Hardness, Disintegration Product Force, lb.f mm % (±0.5%) kp(fragment), 37° C. IL-10 delivery 2,000 4.04 0.0 18.4 2 min 45 sec:swelling + construct erosion 10 min: slight swelling (SEQ ID NO: 2,5003.88 0.0 25.0 initially, then slow erosion 5), F-1 tablets 3,000 3.830.0 26.5 14 mins30 sec: slow erosion From final 3,500 3.80 0.0 27.1 16min: slow erosion blend lot #44A

TABLE 32 F2 tablet properties Friability screen Compression Thickness,(n = 1), % Hardness, Disintegration Product Force, Ib.f mm (±0.5%) kP(fragment), 37° C. IL-10 delivery 2,000 3.74 0.0 14.4 1 min10 sec: rapidconstruct (SEQ collapse to fine ID NO: 5), F-2 particles tablets 2,5003.61 0.0 16.2 10 min: slight swelling From final blend 3 min:erosion Lot#46A 3,000 3.49 0.0 20.4 5 min 30 sec: slow erosion 3,500 3.49 0.0 20.47 min: slow erosion

An additional tablet (F3) was created, which was similar to an F1 tabletbut with glyceryl dibehenate in place of magnesium stearate (TABLE 33).This substitution was made due to incompatibility of magnesium stearatein dissolution of target constructs in solution.

TABLE 33 F3 tablet composition F3 Proportion, Mass per Quantity in g,Phase Constituent Grade % tablet, mg for 15 g batch IG IL-10 delivelyconstruct n/a   8.000% 16 1.2000 SMCC Prosolv SMCC 90  68.000% 13610.2000 Crospovidone Kollidon CL   3.000% 6 0.4500 Glycetyl dibehenateCompritol 888   1.000% 2 0.1500 ATO EG SMCC Prosolv SMCC 90  18.750%37.5 2.8125 Crospovidone Kollidon CL   1.000% 2 0.1500Glycetyldibehenate Compritol 888   0.250% 0.5 0.0375 ATO Total coreweight 100.000% 200 15.0000

Example 13: Evaluation of the In Vivo Performance of Oral CapsuleCoating Formulations in Healthy Subjects

The primary objectives of the study are: to evaluate the in vivoperformance of single oral doses of capsule coating formulations usingscintigraphic methods and to compare the in vivo performance of singleoral doses of a selected capsule coating formulation in the fed andfasted state using scintigraphic methods. The secondary objective of thestudy is: to provide information on the safety of capsule coatingformulations after oral administration. The exploratory objective of thestudy is: to collect fasted pH, temperature and pressure profiles foreach study subject as a SmartPill® transits through the gut.

Study Design

This was a single center, open-label, non-randomized, sequential,single-dose, four-period, scintigraphic imaging study in healthysubjects. A single cohort of 12 subjects was enrolled. Each subjectreceived regimens as outlined in TABLE 34. Capsule coating formulationsapplied to size 0 capsules are described in TABLE 35, and release of theIL-10 delivery construct was predicted to occur in the ileum, proximalcolon (later+1), or distal colon (later+2) Oral capsule coatingformulations were supplied as an enteric-coated size 0 capsule intendedfor oral administration. This study examined capsule coatingformulations and not an IL-10 delivery construct of SEQ ID NO. 5. Thecapsule in this study did not contain any active ingredient. There was a3-day minimum washout period between doses. Following Period 3, therewas a period of interim analysis and review of safety and scintigraphydata from previous periods in order to determine which capsule coatingformulation should be selected to administer in the fed state in Period4.

TABLE 34 Regime administered to healthy patients Period Regimen TestProduct (TP) Dose 1 A Capsule Coating Formulation 1, radiolabelled withNMT 1 MBq ¹¹¹In, in the fasted state 2 B Capsule Coating Formulation 2,radiolabelled with NMT 1 MBq ¹¹¹In, in the fasted state 3 C CapsuleCoating Formulation 3, radiolabelled with NMT 1 MBq ¹¹¹In, in the fastedstate 4 D Capsule Coating Formulation 1, 2 or 3, radiolabelled with NMT1 MBq ¹¹¹In, in the fed state ¹¹¹In: indium-111; NMT: not more than;MBq: megabecquerel

TABLE 35 Coating formulations for scintigraphy study Enteric coatEnteric coat HPMC composition thickness thickness Predicted Number(L30D55/FS30D) (mg) (mg) Release 1 50/50 75 10 Illeum 2 30/70 75 10Later + 1 3 20/80 40 10 Later + 2

Subjects were screened for eligibility to participate in the study up to28 days before dosing in Period 1. For each treatment period, eligiblesubjects were admitted to the clinical unit in the evening on the daybefore dosing (Day 1). Subjects received formulations on the morning ofDay 1 following an overnight fast of a minimum of 10 h (Regimens A, Band C) or following a high-fat breakfast (Regimen D). Administration wasperformed on Day 1 with an appropriate interval between subjects basedon scintigraphic imaging requirements (e.g., approximately 10 min). Thestart time was determined based on logistics. Subjects remained residentin the clinical unit until 24 h post-dose. During each fasted regimen, 4subjects received a SmartPill® Capsule immediately followingadministration of the oral capsule. Each subject received the SmartPillin one regimen only; Subjects 001 to 004 received the SmartPill® inRegimen A, Subjects 005 to 008 received the SmartPill® in Regimen B andSubjects 009 to 012 received the SmartPill® in Regimen C. If data wasstill being captured from a SmartPill® that was in situ, the subject wasnot dosed in the next dosing occasion. There was a minimum washoutperiod of 3 days between each product administration. In all periods, a99mTc-DTPA drink was be administered with each radiolabelled testproduct (TP) to provide an outline of the GI tract in order to enablescintigraphic analysis. A follow-up phone call took place 5 to 7 dayspost-final dose to ensure the ongoing wellbeing of the subjects.

Gamma Scintigraphy

For all regimens, anterior dual isotope images of approximately 50 secduration were collected at regular intervals up to 24 h after dosing.Images from the 99mTc-DTPA radiolabeled drink were used to provide GItract outline only and were not be analysed. Qualitative scintigraphicdata analysis was performed in order to determine the followingparameters: time and anatomical location of initial radiolabel releasefrom the capsule and time and anatomical location of complete radiolabelrelease from the capsule. Anatomical location of radiolabel release wasdefined from the following: stomach, proximal small bowel, distal smallbowel, ascending colon (including the hepatic flexure), transverse colon(including the splenic flexure) and descending colon (including thesigmoid colon and rectum). Qualitative scintigraphic assessment of thetransit of the capsule through the GI tract, prior to completeradiolabel release, was performed by analysing the following parameters:time of gastric emptying and time of colon arrival. Following completeradiolabel release, no further transit parameters were assessed.

pH Telemetry Capsules

During each fasted regimen, four subjects received a SmartPill® Capsuleimmediately after administration of the TP. Each subject received theSmartPill® in one regimen only. The SmartPill® Capsule is a single-useingestible capsule marketed for use by physicians for diagnosticpurposes. Sensors on board an ingestible capsule measure pH, temperatureand pressure as the capsule travels the length of the GI tract.Measurements were transmitted from the capsule within the GI tract viaan amplitude-shift keying modulated radio frequency signal at 434 MHz toa subject-worn Data Receiver and subsequently downloaded to a laptopcomputer for analysis and review once the capsule was passed. MotiliGI™Software performed data analyses automatically and provided to thephysician with a printable report containing gastric emptying time, andmotility index. The SmartPill® Capsule was typically passed within a fewdays.

The shape, size and weight of the coated placebo capsule and SmartPill®were not the same, therefore the 2 capsules may not transit through theGI tract at exactly the same time. However, the SmartPill® provided datathat allow pH, pressure and temperature information from differentregions of the gut to be characterized.

Results

Following administration with capsule coating formulation 1, initialradiolabel release was observed with a mean value of 2.675 h post-dose(TABLES 36, 37; FIG. 32A). Complete radiolabel release was achievedapproximately 1.1 h later, at a mean time of 3.758 h post-dose (TABLES36, 37; FIG. 32B). Anatomical locations of disintegration were noted tobe highly variable, ranging from the stomach to the ascending colon (AC)for initial (FIG. 33A), and to the descending colon (DC) for completerelease (FIG. 33B).

Following administration with capsule coating formulation 2, initialradiolabel release was observed with a mean value of 4.903 h post-dose(TABLES 36, 38; FIG. 32A). Similar to capsule coating formulation 1,complete radiolabel release occurred approximately 1.3 h later, with amean time of 6.176 h post-dose (TABLES 36, 38; FIG. 32B). Anatomicallocations of initial (FIG. 33A) and complete (FIG. 33B) disintegrationdisplayed less variation, with all radiolabel release occurring betweenthe distal small bowel (DSB) and the AC.

Following administration with capsule coating formulation 3, initialradiolabel release occurred at a slightly earlier time than capsulecoating formulation 2 at 4.082 h post-dose (TABLES 36, 39; FIG. 32A).Similar to capsule coating formulations 1 and 2, complete radiolabelrelease was noted to occur approximately 1.2 h later, with a mean timeof 5.308 h post-dose (TABLES 36, 39; FIG. 32B). Anatomical locations ofdisintegration were less variable then those for capsule coatingformulation 1, albeit more variable than those noted for capsule coatingformulation 2. Initial release ranged from the proximal small bowel(PSB) to the AC (FIG. 33A), while complete release ranged from the DSBto the transverse colon (TC) (FIG. 33B).

TABLE 36 Qualitative scintigraphic parameters for the time and locationof initial and complete radiolabel release in healthy volunteersfollowing administration of oral capsule coating formulations. InitialComplete Radiolabel Initial Radiolabel Complete Release * LocationRelease * Location Duration of (h post-dose) Category^(a) (n) (hpost-dose) Category^(a) (n) Disintegration (h) Regimen A 2.675 Stomach(n = 1) 3.758 Stomach (n = 1) 1.083 Capsule Coating (1.140) Stomach/PSB(n = 1) (1.736) PSB (n = 2) (0.857) Formulation 1 PSB (n = 2) DSB (n =5) (N = 12) DSB (n = 5) DSB/AC (n = 1) DSB/AC (n = 1) AC (n = 1) AC (n =2) TC (n = 1) DC (n = 1) Regimen B 4.903 DSB (n = 9) 6.176 DSB (n = 5)1.273 Capsule Coating (1.298) AC (n = 3) (2.289) DSB/AC (n = 2) (1.444)Formulation 2 AC (n = 5) (N = 12) Regimen C 4.082 PSB (n = 1) 5.308 DSB(n = 8) 1.226 Capsule Coating (1.769) DSB (n = 8) (2.738) AC (n = 3)(1.454) Formulation 3 DSB/AC (n = 1) TC (n = 1) (N = 12) AC (n = 2) *Mean (±SD) ^(a)Location categories defined from: Stomach, Proximal smallbowel (PSB), Distal small bowel (DSB), Ascending colon (AC; includingthe hepatic flexure), Transverse colon (TC; including the splenicflexure), Descending colon (DC; including the sigmoid colon and rectum).Where it is not possible to specify the exact location, a joint locationcategory has been used.

TABLE 37 Qualitative scintigraphic parameters in healthy volunteersfollowing administration of oral capsule coating formulation 1 (RegimenA) Anatomical Anatomical Initial position of Complete position ofradiolabel capsule at radiolabel capsule at release time of release timeof Duration Subject (h post- initial (h post- complete of disinte-number dose) release ^(a) dose) release ^(a) gration(h) 001 2.63 PSB4.13 DSB 1.50 002 1.63 DSB 2.88 DSB 1.25 003 2.14 DSB 2.39 DSB 0.25 0042.38 PSB 2.88 PSB 0.50 005 2.13 Stomach 2.38 Stomach 0.25 006 5.13 AC7.88 DC 2.75 007 3.88 AC 4.13 AC 0.25 008 4.13 DSB/AC 5.88 TC 1.75 0091.38 Stom/PSB 1.63 PSB 0.25 010 2.88 DSB 4.13 DSB 1.25 011 1.89 DSB 4.15DSB/AC 2.26 012 1.90 DSB 2.64 DSB 0.74 Mean 2.675 3.758 1.083 SD 1.1401.736 0.857 Median 2.260 3.505 0.995 Min 1.38 1.63 0.25 Max 5.13 7.882.75 n= 12 12 12 ^(a) Location categories defined from: Stomach,Proximal small bowel (PSB), Distal small bowel (DSB), Ascending colon(AC; including the hepatic flexure), Transverse colon (TC; including thesplenic flexure), Descending colon (DC; including the sigmoid colon andrectum). Where it is not possible to specify the exact location, a jointlocation category has been used.

TABLE 38 Qualitative scintigraphic parameters in healthy volunteersfollowing administration of oral capsule coating formulation 2 (RegimenB) Anatomical Anatomical Initial position of Complete position ofradiolabel capsule at radiolabel capsule at release time of release timeof Duration Subject (h post- initial (h post- complete of disinte-number dose) release ^(a) dose) release ^(a) gration (h) 001 5.88 AC6.13 AC 0.25 002 6.88 AC 11.50 AC 4.62 003 3.13 DSB 4.38 AC 1.25 0044.14 DSB 4.40 DSB 0.26 005 6.14 DSB 6.89 DSB 0.75 006 5.88 DSB 9.53DSB/AC 3.65 007 4.38 DSB 5.13 DSB 0.75 008 4.38 DSB 5.13 DSB 0.75 0094.13 DSB 4.38 DSB/AC 0.25 010 3.88 DSB 4.13 DSB 0.25 011 3.39 DSB 5.38AC 1.99 012 6.63 AC 7.13 AC 0.50 Mean 4.903 6.176 1.273 SD 1.298 2.2891.444 Median 4.380 5.255 0.750 Min 3.13 4.13 0.25 Max 6.88 11.50 4.62 n=12 12 12 ^(a) Location categories defined from: Stomach, Proximal smallbowel (PSB), Distal small bowel (DSB), Ascending colon (AC; includingthe hepatic flexure), Transverse colon (TC; including the splenicflexure), Descending colon (DC; including the sigmoid colon and rectum).Where it is not possible to specify the exact location, a joint locationcategory has been used.

TABLE 39 Qualitative scintigraphic parameters in healthy volunteersfollowing administration of oral capsule coating formulation 3 (RegimenC) Anatomical Anatomical Initial position of Complete position ofradiolabel capsule at radiolabel capsule at release time of release timeof Duration Subject (h post- initial (h post- complete of disinte-number dose) release ^(a) dose) release ^(a) gration (h) 001 5.63 DSB5.63 DSB 0.00 002 3.64 DSB 3.88 DSB 0.24 003 8.50 AC 11.50 TC 3.00 0045.38 DSB 8.50 DSB 3.12 005 3.88 DSB 4.63 DSB 0.75 006 3.38 AC 3.63 AC0.25 007 4.15 DSB/AC 8.51 AC 4.36 008 1.88 DSB 3.38 DSB 1.50 009 3.65DSB 4.14 AC 0.49 010 3.88 DSB 4.13 DSB 0.25 011 2.63 PSB 2.88 DSB 0.25012 2.38 DSB 2.88 DSB 0.50 Mean 4.082 5.308 1.226 SD 1.769 2.738 1.454Median 3.765 4.135 0.495 Min 1.88 2.88 0.00 Max 8.50 11.50 4.36 n= 12 1212 ^(a) Location categories defined from: Stomach, Proximal small bowel(PSB), Distal small bowel (DSB), Ascending colon (AC; including thehepatic flexure), Transverse colon (TC; including the splenic flexure),Descending colon (DC; including the sigmoid colon and rectum). Where itis not possible to specify the exact location, a joint location categoryhas been used.

Example 14: Assessment of Efficacy of an IL-10 Delivery Construct in anOxazolone-Induced Mouse Model of Th2 Ulcerative Colitis

The inflammatory bowel diseases (IBD), Crohn's disease (CD) andulcerative colitis (UC), are chronic relapsing disorders characterizedby inflammation of the gastrointestinal (GI) tract. IBD patients sufferfrom progressive and debilitating symptoms resulting from a complexinteraction of genetic contribution, environmental factors and aninappropriate host inflammatory response to luminal antigens, elicitedby the mucosal immune system.

Cytokines control numerous aspects of the immune response involved inestablishing and/or maintaining a proinflammatory or anti-inflammatorybias that control many aspects of health and disease. Along thegastrointestinal (GI) tract, immunomodulatory cells respond to a widevariety of environmental stimuli and are responsible for initiating aproinflammatory signaling cascade in response to pathological antigens.Epithelial cells and cells localized to the underlying lamina propria ofthe GI tract respond to a myriad of cytokines that control theinflammatory status of this tissue. In the case of GI-related IBD,including UC and CD, activation of pro-inflammatory pathways appears tooccur too readily and the resolution of these events, to maintain GIhomeostasis, is insufficient. Thus, the onset, progression, andresolution of IBD conditions are regulated by the balance ofproinflammatory and anti-inflammatory cytokines. By manipulatingGI-associated, proinflammatory cytokines through the actions ofenvironmental insults, it is possible to establish pre-clinical animalmodels that re-create many pathophysiological aspects observed in IBDpatients.

Oxazolone-induced murine colitis represents a reliable system toevaluate potential treatments for IBD where an environmental insult isused to incite an acute inflammatory condition. It is characterized by amixed neutrophil and lymphocyte infiltration limited to the superficiallayer of the mucosa, which is associated with ulceration. In this model,peripheral pre-sensitization is followed by rectal instillation of thehaptenizing agent, oxazolone. This sensitization/activation protocolleads to a Th2-mediated immune response that is marked by an increase intissue interleukin (IL)-4 and IL-5 secretion, reflecting distinguishingmolecular hallmarks of UC. Importantly, oxazolone exposure alsoincreases other tissue cytokines that play a role in inflammation,including the chemokines monocyte chemoattractant protein (MCP)-1,macrophage inflammatory protein (MIP)-1β, growth factorgranulocyte-colony stimulating factor (G-CSF) and the proinflammatorycytokines tumor necrosis factor (TNF)-α and IL-1α. Systemicconcentrations of proinflammatory cytokines and chemokines are generallyalso elevated in IBD patients; specifically, TNF-α secretion maycorrelate with disease severity. Thus, sampling of plasma cytokines canreflect intestinal inflammation and drug efficacy as well as assist inelucidating the mechanism of action for a drug.

The objectives of this study were to: (1) evaluate the ability of anIL-10 delivery construct (SEQ ID NO: 5) solution, delivery by oralgavage, to prevent oxazolone-induced colitis in mice by assessingmultiple-in-life disease parameters (body weight loss, stoolconsistency, hemoccult), survivability, post-necropsy colon morphology(weight and length) and colon histopathology at study termination; (2)assess the anti-inflammatory efficacy and mechanism of the IL-10delivery construct (SEQ ID NO: 5) in isolated intestinal tissue throughimmunohistochemical staining of colonic TNF-α, NFκB, IL-4, CD4 andFoxp3; and (3) assess the anti-inflammatory actions of the IL-10delivery construct (SEQ ID NO: 5) by measuring plasma concentrations ofcytokines and chemokines using the Luminex and Meso Scale Discovery(MSD) platforms.

Methods

Female SJL/J mice were obtained from The Jackson Laboratory, Bar Harbor,Me. 04609 USA. At the commencement of the study, mice were between 7-8weeks of age, weighing 18-22 g. Mice were maintained in a controlledenvironment with a temperature of 70-72° F., humidity 30-70%, and photocycle of 12 hours of light and 12 hours of dark. Mice were provided withTEKLAD 2018-Global 18% diet and Arrowhead drinking water ad libitum.Mice were acclimatized for a period of seven days.

Colitis induction and treatment in mice were conducted by Invitek Inc.(Hayward, Calif.). Mice were pre-sensitized with a 3% oxazolone (SigmaAldrich, USA; catalog #: E0753) in 100% ethanol on a patch of dorsalskin at day −5 and intra-rectally challenged with a 1% oxazolone in 40%ethanol on day 0. Control mice (Naïve) were treated with 100% (day −5)and 40% ethanol (day 0). Mice were treated q.d. by oral gavage (10mL/kg) of the IL-10 delivery construct (SEQ ID NO:5) (8.45 mg/kg),aminosalicylate (5-ASA, 100 mg/kg, dissolved in water; Sigma Aldrich,USA; Catalog #: A3537) or Vehicle control for the IL-10 deliveryconstruct from day −5 through day 6. Experimental design and groupnumbers are summarized in TABLE 40. Daily body weight and diseaseparameters (fecal consistency, hemoccult positivity) were recorded togenerate a disease activity index (DAI). Plasma and colon tissue werecollected at study termination on day 7. Colon weights and lengths weremeasured prior to fixation.

TABLE 40 Experimental design Dose Route of Dose volume Dosing GroupDescription N administration (mg/kg) (ml/kg) frequency 1 Control, no 5p.o. n/a 10 q.d., Day- oxazolone (Naïve) 5-+6 2 Oxazolone 10 p.o. n/a 10q.d., Day- treatment + oral 5-+6 gavage Vehicle (Vehicle) 3 IL-10delivery 15 p.o. 8.45 10 q.d., Day- construct 5-+6 4 5-ASA 15 p.o. 10010 q.d., Day- 5-+6

Hematoxylin and eosin staining of formalin-fixed and paraffin-embeddedcross sections of approximate proximal, mid, and distal colon and theirhistopathology scoring were performed by IDEXX Reference Laboratories,Inc. The presence of colitis and severity score was assessed accordingto the presence of inflammation, leukocyte infiltration, vasculardensity, colon wall thickness, crypt abscesses and the presence ofgoblet cells and ulceration.

Formalin-fixed and paraffin-embedded cross sections of the proximal,mid, and distal colon regions (2 sections each) of treated mice wereprocessed for IHC staining of mouse NF-kB p65, TNF-α, IL-4, CD4, Foxp3by HistoTox (Boulder, Colo.). Image analysis was performed on thedigital slide images using Visiopharm software, using the followingprocedure. The tissues were processed using imaging filters in order toseparate positive staining from counterstaining, then processed imageswere classified using a thresholding method, where a threshold isestablished based on pixel values associated with positively stainedtissues. Quantification of the amount of positive staining wasdetermined by analyzing the labeled image; percent positivity wascalculated by dividing the area of positive tissue by the total tissuearea to provide a metric for positivity.

Plasma cytokines were quantified using the V-plex Proinflammatory Panel1 Mouse Kit. Plasma IL-1Ra was quantitated by sandwich immunoassay usingan antibody pair from the Mouse IL-1Ra/IL-1F3 DuoSet ELISA (R&D Systems#DY480), Streptavidin SULFO-TAG Labeled (MSD #R32AD-1), Multi-Array 96Plate Pack, SECTOR Plate MSD #L15XA-3 and Read Buffer T (4×) (MSD#R92TC-2). Samples were read on the QuickPlex SQ 120 plate reader (MesoScale Discovery, Rockville, Md.).

The Luminex assay was performed in the Human Immune Monitoring Center atStanford University. Mouse 38 plex kits were purchased fromeBiosciences/Affymetrix and used according to the manufacturer'srecommendations with modifications as described herein. Beads were addedto a 96-well plate and washed in a Biotek ELx405 washer. Samples wereadded to the plate containing the mixed antibody-linked beads andincubated at room temperate for 1 hour followed by overnight incubationat 4° C. with shaking. Cold and room temperature incubation steps wereperformed on an orbital shaker at 500-600 rpm. Following the overnightincubation plates were washed in a Biotek ELx405 washer and thenbiotinylated detection antibody added for 75 minutes at room temperaturewith shaking. Plates were washed as above and streptavidin-PE was added.After incubation for 30 minutes at room temperature, washing wasperformed as above and reading buffer was added to the wells. Eachsample was measured in duplicate. Plates were read using a Luminex 200instrument with a lower bound of 50 beads per sample per cytokine.Custom assay control beads by Radix Biosolutions were added to allwells.

Statistical analyses were performed using Prism 5.0 (Invitek data) orPrism 7.0 (GraphPad Software, Inc.). Data were analyzed using one-wayANOVA or two-way ANOVA followed by Bonferroni, Tukey or Dunnett post-hoctests. P values<0.05 were considered significant.

Results

Oxazolone-induced colonic inflammation was associated with a significantreduction in body weight and reduced survivability (Vehicle control vs.Naïve control groups; FIG. 35 and FIG. 36). This decrease in body weightwas found to be attenuated by treatment with 5-ASA (p<0.05 at days 4-7);however no statistical differences were detected in mice receiving theIL-10 delivery construct (FIG. 35). The IL-10 delivery constructtreatment improved survival relative to Vehicle (3% increase), whereas5-ASA treatment led to a more pronounced improvement in survival (10%),(FIG. 36). No differences were detected in stool consistency orhemoccult positivity (presence of blood in stool) between any of thegroups (data not shown). Oxazolone treatment induced colonicinflammation in Vehicle-treated mice, as indicated by thehistopathological severity score, which was attenuated by treatment withthe IL-10 delivery construct (p<0.05; Vehicle vs. IL-10 deliveryconstruct), but not 5-ASA treatment (FIG. 37). Oxazolone treatment alsosignificantly increased colon weight in the Vehicle group, which wasameliorated by the presence of both the IL-10 delivery construct and5-ASA (p<0.01 and p<0.001 respectively), (FIG. 38A). Compared tovehicle-treated animals, improvements for treatment with the deliveryconstruct were observed for hemoccult positivity (FIG. 38B), stoolconsistency (FIG. 38C), and disease activity index (FIG. 38D).Treatment-mediated suppression of increased serum levels of macrophagecolony-stimulating factor 1 (MCSF, FIG. 38E), IL12 p70 protein (FIG.38F), and IL-3 (FIG. 38G) was observed.

NFκB is a transcription factor that plays a chief role in inflammation;signaling through NFκB regulates the proinflammatory cytokines IL-10,IL-6 and TNF-α. To understand the local effect of the IL-10 deliveryconstruct, protein expression of TNF-α, NF-kB, IL-4, CD4, and Foxp3 wasanalyzed in colon sections by IHC (FIGS. 39A-39E). Inflammation inducedby oxazolone (represented by the Vehicle treatment group) led to a trendtowards an increased number of cells in the colon expressing CD4. Thisdata indicated the presence of a greater number of activated CD4+ Tcells, which may include Th2 effector cells, the main driver ofinflammation in this model. Foxp3 expression was also found to beincreased in the Vehicle group (356%, relative to Naïve animals).However, like 5-ASA, treatment with the IL-10 delivery construct did notelicit any significant effect on the expression of any of the proteinsinvestigated.

To further evaluate IL-10 delivery construct efficacy and understand itsmechanisms of action, circulating concentrations of 38 chemokines,growth factors, anti-inflammatory cytokines, and proinflammatorycytokines were determined using the Luminex array. Oxazolone-inducedcolitis (Vehicle group) did not result in statistically-significantchanges in plasma levels of these molecules compared to animals notexposed to oxazolone (Naïve group). However, a trend was observed forincreased plasma levels of the proinflammatory cytokines IL-6 and IL-23(FIGS. 40A-40B) in animals with oxazolone-induced colitis compared withthe Naïve group; plasma levels of these cytokines are elevated in IBD.

Suppression of the trend for oxazolone induction of the proinflammatorycytokines IL-6 and IL-23 was observed with 5-ASA and the IL-10 deliveryconstruct treatment (FIGS. 40A-40B). Compared with Vehicle controls,prophylactic IL-10 delivery construct treatment resulted in plasma IL-23and IL-6 concentrations that were reduced by 73% and 33%, respectively.

The plasma concentration of 10 cytokines and chemokines that play keyeffector or regulator roles in inflammation were determined using thehigh sensitivity of the MSD electrochemiluminescence immunoassay (FIGS.41A-41J). Induction of colitis with oxazolone resulted in trends forincreased levels of all cytokines except for IL-5, although statisticalsignificance was not reached for any (probably on account of the highvariability observed in the Vehicle control groups). Relative to theVehicle controls, the IL-10 delivery construct significantly reduced theexpression of IL-10 and IL-2 (p<0.05) and revealed a trend forsuppressing the induction of IL-4 (62%), IL-6 (61%), IL-12p70 (65%) andthe neutrophil chemoattractant KC/GRO (51%).

Discussion

This in vivo efficacy study evaluated the ability of 8.45 mg/kg IL-10delivery construct (SEQ ID NO: 5) administered by oral gavage to prevent(prophylactic mode of treatment) the development of chemically-inducedcolitis caused by the hapten oxazolone. The IL-10 delivery constructtreatment exhibited efficacy in this model, as evidenced by beneficialeffects on colon weight and histopathological severity score, which arerelevant pathological disease indices established previously with thismodel of induced colitis in mice. Without being bound by any particulartheory, these observations are likely attributable to a direct effect ofthe IL-10 delivery construct on the lamina propria.

At the molecular level, oxazolone increased the number of CD4- andFoxp3-expressing T cells within the colon. However, this model ofoxazolone-induced colonic inflammation did not initiate the significantcascade of chemokines and cytokines generally associated with the immuneresponse in UC. The absence of a robust response may be due to thepresence of high variability within the treatment groups.

Conclusion

In conclusion, the results collectively demonstrated that prophylactictreatment with the IL-10 delivery construct (SEQ ID NO: 5), delivered asan 8.45 mg/kg (mpk) oral dosing solution, had the capacity to suppressoxazolone-induced colitis in mice, as assessed by clinical signs of UC.While oral delivery of the IL-10 delivery construct (SEQ ID NO: 5) hadno clear effect on the immunological expression profile within thecolon, subchronic dosing did elicit significant and trending suppressionof systemic proinflammatory cytokines that are associated withintestinal inflammation.

Example 15—Dose-Response Efficacy of an IL-10 Delivery Construct in theMurine Models of Oxazolone- and Dextran Sulfate Sodium (DSS)-InducedUlcerative Colitis

The DSS-induced experimental colitis model in mice employs the deliveryof a chemical colitogen with anticoagulant properties in the drinkingwater. The insult results in damage to the epithelial monolayer of thelarge intestine and the dispersal of pro-inflammatory intestinalcontents into the underlying tissue. The popularity of this model in IBDresearch arises from its rapidity and reproducibility, and the model canbe manipulated to elicit acute, chronic and relapsing models of IBDdepending on the concentration of DSS and the frequency ofadministration.

Example 13 demonstrated the therapeutic potential of the IL-10 deliveryconstruct when dosed prophylactically; an oral dosing solution of 8.45mg/kg IL-10 delivery construct showed moderate efficacy for preventingcolitis development as measured by multiple disease parameters.

In this Example, both the oxazolone and DSS-induced colitis models wereused to evaluate the dose-response efficacy of the IL-10 deliveryconstruct (SEQ ID NO: 5). The anti-inflammatory agent, 5-ASA, wasincluded within the study to serve as a positive control for suppressionof oxazolone-induced inflammation. 5-ASA has been widely used in theclinic to treat mild to moderate UC due to its relative effectiveness,safety and high tolerability. The immunosuppressant cyclosporine (CsA)was utilized as a positive control in the DSS study; this treatment isfrequently used in the clinic for the treatment of Crohn's disease (CD).

To evaluate target engagement of our dosing solution, the IL-1 receptorantagonist (IL-1Ra) was evaluated as a candidate pharmacodynamicbiomarker of the IL-10 delivery construct. Secreted by epithelial,immune cells and adipocytes, IL-1Ra binds the IL-1 receptor but fails toinduce signaling, thus antagonizes IL-1-mediated inflammation. Theefficacy of IL-10 delivery construct prophylactic treatment was assessedby evaluating a number of disease parameters in vivo. In addition, theeffect of the IL-10 delivery construct on circulating concentrations ofcytokines and chemokines that are altered with intestinal inflammationwas also assessed.

The objectives of this study were to: (1) evaluate the dose-response tothe IL-10 delivery construct (0.3, 1, 3 and 9 mg/kg) inoxazolone-induced murine colitis by assessing multiple in-life diseaseparameters (body weight loss, stool consistency, hemoccult),survivability, post-necropsy colon morphology (weight and length) andcolon histopathology; (2) evaluate the dose response to the IL-10delivery construct (0.3, 3, 10, 30 mg/kg) in the DSS-colitis model byassessing in-life and necropsy disease parameters and histology; (3)assess the anti-inflammatory efficacy and mechanism of the IL-10delivery construct by measuring the plasma concentrations of cytokinesand chemokines using the Luminex and MSD platforms; and (4) assesspotential IL-10 delivery construct-induced pharmacodynamic biomarkers bydetermining tissue gene expression and plasma concentrations of IL-1Raand tissue concentration of pSTAT3.

Methods

Mice were maintained in a controlled environment with a temperature of70-72° F., humidity 30-70%, and photo cycle of 12 hours of light and 12hours of dark. Mice were provided with TEKLAD 2018-Global 18% diet andArrowhead drinking water ad libitum. Mice were acclimatized for a periodof seven days.

Colitis induction by oxazolone and prophylactic treatment in mice wereconducted. Female SJL/J mice between 7-8 weeks of age werepre-sensitized with a 3% solution of oxazolone in 100% ethanol (SigmaAldrich, USA; Catalog #: E0753) on a patch of dorsal skin at day −5 andintra-rectally challenged with a 1% oxazolone solution in 40% ethanol onday 0. The animals were orally treated q.d. with dosing solutions of theIL-10 delivery construct (SEQ ID NO: 5) (0.3, 1, 3, and 9 mg/kg),aminosalicylate (5-ASA, 100 mg/kg; Sigma Aldrich, USA; Catalog #: A3537)or Vehicle control (10 mg/mL soybean trypsin inhibitor in 200 mM sodiumbicarbonate; IL-10 delivery construct formulation buffer) from day −5through day 6. Naïve mice were pre-sensitized and challenged withethanol alone. Daily body weight and disease parameters (fecalconsistency, hemoccult positivity) were recorded to generate a diseaseactivity index (DAI). Plasma and colonic tissues were collected at studytermination on day 7. This study was performed under non-GLP conditionsand conducted according to the INVITEK protocol and INVITEK StandardOperating Procedures. Experimental design and group numbers for theoxazolone treatment are summarized in TABLE 41.

DSS-induction of murine colitis was performed by Bolder BioPATH, Inc.(CO, USA). Female C57BL6/J mice (8-10 weeks old) were given 2.5 dextransodium sulfate (w/v) (Spectrum, Lot #2DC0020) ad libitum in the drinkingwater from day 0 through day 7. On day 7, DSS was replaced with water,and the animals were maintained until day 10. The mice were orallyadministered (q.d.) the IL-10 delivery construct (0.3, 3.0, 10 or 30mg/kg), Vehicle control (10 mg/mL soybean trypsin inhibitor in 200 mMsodium bicarbonate; formulation buffer for the IL-1 delivery construct)or a positive control cyclosporine A (Teva, Lot #4R506001) which wasprepared in Kolliphor EL (Sigma, C5135, Lot #BCBP4773V) and 100carboxymethylcellulose (CMC: BBP, Batch #2017, Lot #3) at 75 mg/kgbeginning day 0 until day 10, when they were sacrificed for plasma andtissue collection at 4 hours post-dose. Experimental design and groupnumbers for the DSS treatment are summarized in TABLE 42.

TABLE 41 Oxazolone group and treatment information Dose Route of Dosevolume Dosing Group Description N administration (mg/kg) (ml/kg)frequency A Control, no 5 p.o. N/A 10 q.d., Day- oxazolone 5-+6 (Naïve)B Oxazolone + oral 10 p.o. N/A 10 q.d., Day- gavage Vehicle 5-+6(Vehicle) C 5-ASA (positive 15 p.o. 100 10 q.d., Day- control) 5-+6 DIL-10 delivery 15 p.o. N/A 10 q.d., Day- construct (9.0 5-+6 mg/kg;Blue) E IL-10 delivery 15 p.o. N/A 10 q.d., Day- construct (3.0 5-+6mg/kg; Green) F IL-10 delivery 15 p.o. N/A 10 q.d., Day- construct (1.05-+6 mg/kg; Red) G IL-10 delivery 15 p.o. N/A 10 q.d., Day- construct(0.3 5-+6 mg/kg; Purple)

TABLE 42 DSS group and treatment information Dose Route of Dose volumeDosing Group Description N administration (mg/kg) (ml/kg) frequency 1Naïve 5 n/a n/a n/a n/a 2 Vehicle 10 PO n/a 10 q.d., Day 0-10 3 CsA 10PO 75 10 q.d., Day 0-10 4 IL-10 delivery 10 PO 0.3 10 q.d., Dayconstruct 0-10 5 IL-10 delivery 10 PO 3 10 q.d., Day construct 0-10 6IL-10 delivery 10 PO 10 10 q.d., Day construct 0-10 7 IL-10 delivery 10PO 30 10 q.d., Day construct 0-10

For both the oxazolone and DSS colitis studies, hematoxylin and eosinstaining of formalin-fixed and paraffin-embedded cross sections of theapproximate proximal, mid, and distal colon were conducted by BolderBioPath (Boulder, Colo.). For each region, two equidistant pieces arecut and embedded in paraffin. Each piece was evaluated individually, andvalues are averaged separately for the proximal, middle, distal, andtotal colon. Histopathology was blindly assessed by the presence ofedema, inflammation, gland loss, erosion, mucosal thickness andhyperplasia to give a summed score. The presence of inflammatory cellinfiltrates and lymphoid aggregate count and diameter were alsodetermined.

The Luminex assay was performed in the Human Immune Monitoring Center atStanford University. Mouse 38 plex kits were purchased fromeBiosciences/Affymetrix and used according to the manufacturer'srecommendations with modifications as described herein. Beads were addedto a 96-well plate and washed in a Biotek ELx405 washer. Samples wereadded to the plate containing the mixed antibody-linked beads andincubated at room temperate for 1 hour followed by overnight incubationat 4° C. with shaking. Cold and room temperature incubation steps wereperformed on an orbital shaker at 500-600 rpm. Following the overnightincubation, plates were washed in a Biotek ELx405 washer and thenbiotinylated detection antibody added for 75 minutes at room temperaturewith shaking. Plates were washed as above and streptavidin-PE was added.After incubation for 30 minutes at room temperature, washing wasperformed as above and reading buffer was added to the wells. Eachsample was measured in duplicate. Plates were read using a Luminex 200instrument with a lower bound of 50 beads per sample per cytokine.Custom assay control beads by Radix Biosolutions were added to allwells.

RNA was extracted from the near-distal colon segments (between 1-2 cmfrom the rectum) of oxazolone-inflamed and treated mice. RNA purity wasconfirmed with absorbance ratios at 260/280 of 1.9-2.15 and 260/230>1.7.RNA samples were reverse transcribed to cDNA using RNeasy Mini Kit(Qiagen, #74106) and iScript™ cDNA Synthesis Kit (Bio-Rad, #1708891BUN).RT-PCR analysis of mouse IL-1Ra, IL-10 and GAPDH was performed intechnical duplicates using Applied Biosystems PowerUp SYBR Green MasterMix (Thermo Fisher Scientific, #A25777). Primer sequences are listed inTABLE 43. Transcript expression was normalized to that of an internalcontrol, GAPDH, and fold-changes were calculated using the AA-CT method.

TABLE 43 RT-PCR primer sequences Primer Name Sequence SEQ ID NO:ms IL-1Ra-F GCTCATTGCTGGGTACTTACAA SEQ ID NO: 14 ms IL-1Ra-RCCAGACTTGGCACAAGACAGG SEQ ID NO: 15 ms IL-1β-F CACAGCAGCACATCAACAAGSEQ ID NO: 16 ms IL-1β-R GTGCTCATGTCCTCATCCTG SEQ ID NO: 17 ms GAPDH-FTGTGTCCGTCGTGGATCTGA SEQ ID NO: 18 ms GAPDH-R CCTGCTTCACCACCTTCTTGASEQ ID NO: 19

Plasma cytokines were quantified using the V-plex Proinflammatory Panel1 Mouse Kit. Plasma IL-1Ra was quantitated by sandwich immunoassay usingan antibody pair from the Mouse IL-1Ra/IL-1F3 DuoSet ELISA (R&D Systems#DY480), Streptavidin SULFO-TAG Labeled (MSD #R32AD-1), Multi-Array 96Plate Pack, SECTOR Plate MSD #L15XA-3 and Read Buffer T (4×) (MSD#R92TC-2). Samples were read on the QuickPlex SQ 120 plate reader (MesoScale Discovery, Rockville, Md.).

The Human IL-10 Base kit (MSD #K151AOA-4) was used to measure rhIL-10 inplasma. The anti-human IL-10 capture and detection antibody pair in thisimmunoassay did not react with mouse IL-10, but did react with humanIL-10. The MSD Small Spot IL-10 plate was incubated with Diluent 41 (25μL/well) for 30 minutes at room temperature with shaking before use.Controls and samples were diluted 2-fold with pooled CD-1 mouse plasma(BioIVT, custom order), and the calibration standards were also preparedin mouse plasma. Standards, diluted samples and controls were added (25μL/well), and the plate was incubated for 2 hours at room temperaturewith shaking. The plate was washed 3 times with phosphate bufferedsaline with Tween-20 (PBST), and 25 μL/well IX SULFO TAG Anti-humanIL-10 Antibody (prepared in Diluent 45) was added to the plate. Theplate was incubated for 1 hour at room temperature with shaking.Following a wash step, 150 μL/well of 2×MSD Read buffer was added, andthe plate was read in the MSD Sector Imager 600 plate reader.

Statistical analyses were performed using Prism 5.0 (Invitek data) orPrism 7.0 (GraphPad Software, Inc.). For the oxazolone study, data wereanalyzed using one-way ANOVA or two-way ANOVA followed by Bonferroni,Dunnett, or Tukey post-hoc tests. DSS-data were analyzed using one-wayANOVA followed by Dunnett's post-hoc test for parametric data or theKruskal-Wallis test with Dunn's post-hoc test for non-parametric data. Pvalues of <0.05 were considered significant.

Results: Oxazolone-Induced Colitis

Oxazolone-induced colonic inflammation led to pronounced body weightloss (up to 15%) and reduced survivability (FIG. 42 and FIG. 43). Theanti-inflammatory agent, 5-ASA, significantly attenuated theoxazolone-induced decrease in body weight from 6 days after the insult(p<0.05). The IL-10 delivery construct at the highest concentrations (1,3 and 9 mg/kg) produced ameliorating trends in body weight loss. Nosignificant effects on DAI, reflecting fecal consistency and hemoccultpositivity, were detected between the treatment groups (FIG. 44).

In addition, oxazolone induced a reduction in colon length and anincrease in colon weight, as reflected by an increase in the colonweight/length ratio (FIG. 45). This increase was ameliorated bytreatment with 5-ASA; however, IL-10 delivery construct had nostatistically significant effect on colon weight or length at any of thedoses tested.

Seven inflammatory parameters (inflammation of mucosa/submucosa,erosion, gland loss, hyperplasia, edema, transmural inflammation,inflammation of serosa) were scored on a scale of 1-4 (1=minimal,2=mild, 3=moderate, 4=marked) to assess the histopathology of theproximal, mid, and distal colon. Based on the summed score (histologyscore) of these seven parameters, histopathology was found to be mostpronounced in the distal colon and least in the proximal colon for alltreatment groups (FIG. 46). The IL-10 delivery construct, at all dosestested, did not induce any changes in histology score in the proximal,mid or distal colon, when compared to the Vehicle control.

In addition to gross disease parameters, IL-10 delivery constructefficacy was also evaluated by assessing the circulating concentrationsof 38 growth factors, chemokines and cytokines (FIGS. 47A-47LL).Oxazolone treatment induced an increase in the plasma concentration ofgranulocyte colony-stimulating factor (GCSF, also known ascolony-stimulating factor 3, or CSF3; FIG. 47A) and a trend towards anincrease in the macrophage chemoattractant MIP1α (FIG. 47L) and thegrowth factors granulocyte-macrophage colony-stimulating factor (GMCSF;FIG. 47B) and macrophage colony stimulating-factor (MCSF;

FIG. 47C), which respectively mobilize the release and differentiationof neutrophils and macrophages. Prophylactic pre-treatment with thepositive control, 5-ASA, or the IL-10 delivery construct attenuated theoxazolone-induced increase in GCSF/CSF3. MSCF was also reduced at thehighest dose of the IL-10 delivery construct. A trend towards areduction was also observed for MIP1α (FIG. 47L). Changes inLPS-inducible CXC chemokine (LIX, FIG. 47I), leukemia inhibitory factor(LIF, FIG. 47E), and IP10 (CXCL10, FIG. 47H) levels induced by Oxa weremitigated by treatment with the IL-10 delivery construct.

In addition to affecting innate immune cells, cytokines also controleffector T cell functions. Consistent with an anti-inflammatory effect,IL-10 delivery construct pre-treatment (9.0 mg/kg dose) prevented theproduction of IL-12 and IL-17, key effectors of Th1 and Th17 cells,respectively. Treatment with the IL-10 delivery construct led to areduction in the concentrations of IL-3 (9.0 mg/kg dose; FIG. 47S), IL-4(9.0 mg/kg; FIG. 47T), IL-28 (3.0 and 9.0 mg/kg; FIG. 47EE), and IL-31(0.3 mg/kg; FIG. 47FF) to near-base line levels. Plasma concentrationsof IL-13 (FIG. 47Y) and IL-15 (FIG. 47Z) revealed a trend towards anIL-10 delivery construct-mediated decrease, compared with the Vehiclecontrol. Beyond the pro-inflammatory cytokines, oxazolone (Vehicletreatment alone) indicated a trend towards increased secretion ofendogenous IL-10 (FIG. 47JJ) and the immune-suppressive cytokine TGF-β(FIG. 47LL). This apparent counter-response to inflammation isconsistent with the observed increase of TGF-β in UC colon tissue. IL-10delivery construct treatment (3.0 mg/kg) reduced TGF-β relative toVehicle and ameliorated this trend towards increased secretion ofendogenous IL-10. For many of the cytokines analyzed, plasmaconcentration was not significantly induced following oxazolonetreatment. Moreover, when an oxazolone-induced increase was observed,5-ASA did not effectively return the expression of the cytokine tobaseline (Naive) levels in many of the parameters tested.

Systemic cytokine concentrations in mice exposed to oxazolone-inducedcolitis and prophylactic treatment with the IL-10 delivery constructwere additionally assessed using the MSD 10-plex proinflammatory panel.Plasma IFN-γ and IL-5 were reduced in Vehicle-treated mice withoxazolone-induced colitis vs Naïve controls, but there were nosignificant differences between these groups with respect to the othercytokines measured (FIGS. 48A-48J). Trend for increases in theexpression of IL-10 and IL-6 were observed in response to the oxazoloneinsult. However, this response was not attenuated by the delivery of thepositive control, 5-ASA, or the IL-10 delivery construct. Due to theabsence of a substantial oxazolone-induced inflammatory response, theeffect of the IL-10 delivery construct treatment was not easilydecipherable; however, no significant differences between Vehicletreatment and IL-10 delivery construct were detected for any of thecytokines analyzed.

IL-10 delivery construct efficacy in the systemic circulation andcolonic tissue was assessed by analyzing the expression of IL-1Ra, anIL-10 antagonist that is potentiated by IL-10. Circulating levels ofIL-1Ra were not significantly altered following oxazolone insult ortreatment with 5-ASA or the IL-10 delivery construct (FIG. 49A), likelydue to data variability and low statistical power. However, the IL-10delivery construct treatment did induce a dose-dependent trend towardsincreased IL-1Ra. In near-distal colonic tissue, the IL-10 deliveryconstruct treatment at the highest dose of 9 mg/kg revealed a trendtowards augmented mRNA expression of IL-1Ra (FIG. 49B). This result wasalso reflected by an increase in the IL-1Ra/IL-10 ratio (FIG. 49D).

Results: Dextran Sulfate Sodium (DSS)-Induced Colitis

This example examines the IL-10 delivery construct's ability to suppressthe mild to moderate multifocal colitis induced by dextran sulfatesodium (DSS), characterized by inflammation, edema, and mucosal necrosiswhen this medium-chain-length fatty acid binding agent is presented indrinking water. Mice were dosed orally once daily for 10 days withvehicle (as a negative control) or the IL-10 delivery construct of SEQID NO: 5 (0.3-30 mg/kg) from the induction of DSS-induced colitis (FIG.50A). Mice dosed daily with 75 mg/kg cyclosporine A (CsA) were used as apositive control for the model and not a therapeutic comparator. Thedelivery construct of SEQ ID NO: 5 significantly reduced DSS-inducedweight loss during the in-life portion of the study (FIG. 50B). DSSinsult led to ˜10% weight loss in the Vehicle group (or ˜17%differential with the Naïve group, which gained weight), (FIG. 50C).Treatment with CsA partially attenuated the DSS-induced loss in bodyweight (p<0.05), as was observed with the IL-10 delivery construct, atthe highest dose tested (30 mg/kg). There was no mortality in any of thetreatment groups. Individual scores for weight loss, stool consistencyand stool hemoccult (scored 0-3) were also summed to provide a DAI (0-9range). Relative to the summed score of 3.8 for the Vehicle group on day7 (and 0 for the Naïve group), scores for IL-10 deliveryconstruct-treated groups ranged from 4.0 to 4.3 withoutdose-correlation, and thus do not support disease activity index (DAI)improvement by the IL-10 delivery construct (scores were not availablefor day 10), (FIG. 51). Significant improvement in colon length at thetwo top doses of the IL-10 delivery construct (10 and 30 mg/kg; p<0.05compared to vehicle) indicate the presence of IL-10 deliveryconstruct-dependent therapeutic effects (FIG. 52).

Colon histology was scored based on inflammation, gland loss, erosionand hyperplasia (max of 5 each), and these were totaled in the summaryscore. All four parameters were significantly increased following theDSS insult in the middle and distal colon (p<0.05 vs. Naïve). CsAtreatment alleviated the summed score of the four parameters measured,but treatment with the IL-10 delivery construct did not reveal anysignificant improvements (FIG. 53). Five additional parameters (edema,neutrophil infiltration, mucosal thickness, lymphoid aggregate andlymphoid aggregate size) were also quantified. Consistent with thepropagation of disease from the distal to proximal colon, histopathologywas most severe in the distal colon and negligible in the proximal colonwith Vehicle treatment for several parameters. Relative to Vehicle, theIL-10 delivery construct (30 mg/kg) treatment significantly improvededema width (FIG. 54), mucosal thickness (FIG. 55), and hyperplasia(FIG. 56) in the mid region. CsA did not show any clear therapeuticeffects following DSS insult, with the exception of an improvement inneutrophil score (p<0.05 vs. Vehicle). These histological results thusindicate a mild degree of local drug efficacy.

IL-10 delivery construct was detected at variable concentrations in thecirculation 4 hours after the final dose in the DSS study. Levels ofrhIL-10 (detected by anti-rhIL-10 capture antibody) ranged from 10-300fold higher than the background concentrations of Vehicle treatment, inanimals treated with the IL-10 delivery construct at doses of 0.3 and 3mg/kg (FIG. 57A); however, this effect is lost with higher IL-10delivery construct dosing. Furthermore, at the low IL-10 deliveryconstruct doses, higher concentrations of rhIL-10 than IL-10 deliveryconstruct (determined with the anti-cholix capture antibody) indicatethe presence of IL-10 delivery construct cleavage products, which wouldbe consistent with protease activities prevalent in the GI environment.Prophylactic treatment with the IL-10 delivery construct did not appearto influence plasma concentrations of IL-1Ra at any dose tested (FIG.57B).

Discussion

In these studies, the efficacy of an IL-10 delivery construct (SEQ IDNO: 5) for preventing the development of inflammatory colitis in theoxazolone and DSS models was evaluated. In both studies, trends forimprovement in a number of in-life and necropsy parameters wereobserved. Improvement in colon length at the two highest doses of theIL-10 delivery construct (10 and 30 mg/kg) following the DSS insultprovided evidence for IL-10 delivery construct-dependent therapeuticeffects. No mortality was present in the DSS study, whereas survival wasreduced by up to 30% following oxazolone treatment, suggesting that theoxazolone protocol induced a much more severe model of colitis.

A notable feature of the DSS model was that the histopathology was moreconsistent between biological replicates; intra-group variabilityappeared to be reduced in the DSS model compared with the oxazolonemodel. In the DSS model, the IL-10 delivery construct at the highestdose (30 mg/kg) effectively reduced three histology parameters, namelyedema width, mucosal thickness and hyperplasia, in the mid but notdistal colon.

In the presented dose-response oxazolone study, efficacy wasdemonstrated by changes in circulating cytokine concentrations mediatedby the IL-10 delivery construct. The plasma concentration of GCSF (orCSF3) was increased in response to the oxazolone insult, and thisresponse was mitigated by prophylactic pre-treatment with 5-ASA or theIL-10 delivery construct. Pre-treatment with the IL-10 deliveryconstruct (9.0 mg/kg dose) also prevented the production of IL-12 andIL-17, key effectors of Th1 and Th17 cells. Additionally, the IL-10delivery construct led to a reduction in the concentrations of MCSF (9.0mg/kg dose), IL-3 (9.0 mg/kg), IL-4 (9.0 mg/kg), IL-28 (3.0 mg/kg and9.0 mg/kg), and IL-31 (0.3 mg/kg) to near basal (Naïve) levels. Plasmaconcentration for many of the cytokines analyzed were not significantlyaltered following oxazolone treatment. When an oxazolone-inducedincrease was present, the positive control 5-ASA did not effectivelyreturn the expression of the cytokine to baseline levels in many of theparameters tested. Thus, in the absence of a robust attenuation ofinflammatory cytokines by 5-ASA, the suitability of this agent as acontrol treatment may be questioned. Additionally, this model ofoxazolone-induced inflammation may not be sufficient to trigger asubstantial systemic cytokine response in these mice.

IL-10 delivery construct treatment at the highest dose of 9 mg/kgrevealed a trend towards augmented mRNA expression of IL-1Ra and a trendtowards an increase in the IL-1Ra/IL-10 ratio in colonic tissue.Previous clinical data has demonstrated that the ratio of IL-1Ra/IL-10inversely correlates with IBD severity, and that IL-10 can restore theIL-1Ra/IL-13 ratio. These results provide evidence for the IL-10delivery construct treatment efficacy in this model.

Conclusion

To conclude, in the context of both oxazolone- and DSS-inducedinflammatory colitis, the data presented in this report indicated thattreatment with the IL-10 delivery construct (SEQ ID NO: 5) demonstratedtherapeutic efficacy, as measured by improvements in inflammatoryparameters in vivo. In the oxazolone study, circulating concentrationsof IL-12, IL-17 and IL-28 were attenuated by treatment with the IL-10delivery construct, which was accompanied by a trend towards an increasein the IL-1Ra/IL-10 ratio in colonic tissue.

Example 16—Oral Administration of a Single-Dose of IL-10 DeliveryConstruct in Macaca fascicularis

The non-human primate (NHP), Macaca fascicularis, was selected as amodel system to test evaluate the preclinical efficacy, pharmacologicalactivity and pharmacokinetics of an IL-10 delivery construct (SEQ ID NO:5). Due to the significant genetic similarity between humans and NHPssuch as the Macaca fascicularis, the NHP generally represents a moreappropriate model for humans than the mouse, as the NHPs more closelymimic human biology and immunology. The addition of 10 mg caffeine tothe IL-10 delivery construct capsules was utilized in this study toinvestigate capsule opening.

The objectives of this study were to: (1) evaluate the impact of singledosing at multiple levels (1, 4, 20.5 and 82 mg) by oral capsule on thepharmacokinetic profile of the IL-10 delivery construct (as measured bytotal IL-10 and established biomarker IL-1Ra) in Macaca fascicularis;(2) assess the response of the pro-inflammatory cytokines IFNγ, IL-10,IL-2, IL-6, IL-8 and TNFα to a single oral dose at multiple levels (1,4, 20.5 and 82 mg) of the IL-10 delivery construct by capsule in Macacafascicularis; and (3) investigate the capsule performance by monitoringplasma levels of caffeine in dosed Macaca fascicularis, as included inthe aforementioned orally dosed capsules.

Methods

IL-10 delivery constructs (SEQ ID NO: 5) and caffeine were filled byweight into size-1 Capsugel V-Cap Hydroxypropyl Methylcellulose (HPMC)capsules, for oral administration. The capsules were then successivelycoated in a pan spray coater with three layers of polymers. The firstand third layer were thin coatings of HPMC, the first to seal the creasewhere the capsule shells come together, and the third to minimizesticking of the capsules to each other. The second coating layer wascomprised of a 50:50 mixture of Eudragit® (Evonik Industries AG) entericacrylic polymers FS 30 D and L 30 D-55, designed to dissolve and allowthe capsule to open at pH 6.5. A summary of the test articles described,and their corresponding lot numbers is presented in TABLE 44.

TABLE 44 Summary of test articles Name Description 1 mg Size 1 entericcoated capsule containing 1 mg IL-10 IL-10 delivery delivery construct(SEQ ID NO: 5) + 10 mg caffeine construct capsule 20.5 mg Size 1 entericcoated capsule containing 20.5 mg IL-10 IL-10 delivery deliveryconstruct (SEQ ID NO: 5) + 10 mg caffeine construct capsule

The MSD Small Spot IL-10 plate was washed 3 times with PBST before use.Standards were prepared in Diluent 2 (MSD Cat No R51BB), and samplesdiluted 2-fold in Diluent 2. Standards and diluted samples were added tothe assay plate and the plate incubated for 2 hours at room temperaturewith shaking. The plate was washed 3 times with PBST and 1×SULFO TAGanti-huIL-10. Antibody was added to the plate before incubation for 1hour at room temperature with shaking. Following a wash step, 2×MSD Readbuffer was added to the wells of the plate and the plate is read in theMSD Sector Imager 600 plate reader. The lower limit of detection of eachanalyte is defined as 2.5 standard deviation above background.

The purpose of the IL-1Ra assay was to measure endogenous IL-1Ra in NHPplasma. The antibody pair in this sandwich immunoassay reacted with bothhuman and NHP IL-1Ra. Samples were quantitated using an 8-point standardcurve prepared from a human IL-1Ra calibrator (ranging from 5.5-4021μg/mL, plus 0 μg/mL). Biotinylated anti-NHP IL-1Ra capture antibody wasadded to a MSD Small Spot Streptavidin plate and incubated for an hourat RT with shaking. Controls that represent low (16 mg/mL), mid (160mg/mL) and high concentrations (3245 μg/mL) were prepared in pooledplasma, Standards (Calibrator 9 Blend) were prepared in Diluent 43 (MSDCat No R50AG), and samples diluted 10 fold in Diluent 43. Standards,controls and diluted samples were added to the assay plate, and theplate incubated for 2 hours at room temperature with shaking. The platewas washed 3 times with PBST, and 1×SULFO TAG anti-IL-1Ra Antibody addedto the plate. The plate was incubated for 1 hour at room temperaturewith shaking. Following a wash step, 2×MSD Read buffer was added to thewells of the plate, and the plate read using the MSD Sector Imager 600plate reader. The lower limit of detection of each analyte is defined as2.5 standard deviation above background.

In order to quantitate plasma caffeine levels, an LC-MS/MS detectionsystem was employed. A 25-μL matrix aliquot was fortified with 25 μL of2.00 μg/mL caffeine-trimethyl-¹³C₃ internal standard working solution.Analytes were isolated through supported liquid extraction (SLE). Aportion of the eluate was transferred and evaporated under a nitrogenstream at approximately 45° C., and the remaining residue reconstitutedwith 500 μL of water/acetonitrile (95:5, v/v). The final extract wasanalyzed via HPLC with MS/MS detection using positive ion electrosprayand reverse phase chromatography. Caffeine concentration was determinedusing similarly run calibration controls.

The panel of cytokines (IFNγ, IL-10, IL-2, IL-6, and IL-8) was evaluatedusing a multiplex kit available from MSD. The assays in this panel weresandwich immunoassays. The MSD plate came precoated with captureantibodies on independent and pre-defined spots, and samples werequantitated against an 8-point standard curve prepared using a mixtureof the cytokines (Proinflammatory Panel 1 (human) Calibrator Blend) fromMSD. The standards included 0 μg/mL and these concentration ranges: IFNγ(0.37-1500 μg/mL), IL-10 (0.14-589 μg/mL), IL-2 (0.36-1490 μg/mL), IL-6(0.18-721 μg/mL) and IL-8 (0.14-553 μg/mL).

The MSD plate (pre-coated with capture antibodies) was washed 3 timeswith PBST before use. Standards were prepared in Diluent 2 (MSD Cat NoR51BB) and test samples diluted 2 fold in Diluent 2. Standards anddiluted samples were added to the assay plate, and the plate incubatedfor 2 hours at room temperature with shaking. The plate was washed 3times with PBST, and a mixture of 1×SULFO TAG detection antibodies addedto the plate. The plate was incubated for 2 hours at room temperaturewith shaking. Following a wash step, 2×MSD Read buffer was added to thewells of the plate, and the plate read using the MSD Sector Imager 600plate reader. The lower limit of detection of each analyte was definedas 2.5 standard deviation above background.

The purpose of the TNFα immunoassay was to quantify NHP TNFαconcentration in plasma. The antibody pair in this immunoassay reactedwith both human and NHP TNFα. Samples were quantitated using an 8-pointstandard curve prepared from a human TNFα calibrator that ranges inconcentration from 5.6-4052 μg/mL and includes 0 μg/mL.

The biotinylated anti-NHP TNFα capture antibody was added to a MSD SmallSpot Streptavidin plate and incubated for an hour at RT with shaking.Controls were prepared in pooled plasma, standards in Diluent 43 (MSDCat No R50AG), and test samples were diluted 2 fold in Diluent 43.Standards, controls and diluted samples were added to the assay plate,and the plate incubated for 2 hours at room temperature with shaking.The plate was washed 3 times with PBST, and 1×SULFO TAG anti-huTNFαAntibody added to the plate. The plate was incubated for 1 hour at roomtemperature with shaking. Following a wash step, 2×MSD Read buffer wasadded to the wells of the plate, and the plate read using the MSD SectorImager 600 plate reader. The lower limit of detection of each analytewas defined as 2.5 standard deviation above background.

Plasma samples were analyzed for Total IL-10, IL-1Ra, caffeine contentand a panel of proinflammatory cytokines. Assay LLOQs were: totalIL-10=0.3 μg/mL; IL-1Ra=78 μg/mL; and caffeine=25 ng;mL. Plasmaconcentration of IL-10 can refer to the concentration in plasma of bothnaturally occurring IL-10 and IL-10 appended to a carrier (for example,IL-10 in an IL-10 delivery construct described herein).

IL-10 delivery construct dosing at multiple fixed doses in non-naïveadult male Macaca fascicularis was conducted by Valley Biosystems (WestSacramento, Calif.). Adult NHPs were orally treated with capsulescontaining a fixed dose of the IL-10 delivery construct (SEQ ID NO: 5)and 10 mg caffeine (1, 4, 20.5 and 82 mg). Dosing groups are describedin TABLE 45.

TABLE 45 Summary of dose groups SEQ ID NO: 5 Dose per Caffeine Group NRoute Test Article Dose animal Dose A 6 per Oral IL-10 delivery 20.5 mgOne 10 mg group gavage construct + capsule of 10 mg caffeine 20.5 mg B 6per Oral IL-10 delivery 82 mg Four 40 mg group gavage construct +capsules of 10 mg caffeine 20.5 mg C 6 per Oral IL-10 delivery 1 mg One10 mg group gavage construct + capsule of 10 mg caffeine 1 mg D 6 perOral IL-10 delivery 4 mg Four 40 mg group gavage construct + capsules of10 mg caffeine 1 mg

Animals were fasted until after the 3 hour blood sample collection, whenfood was given. For dosing, the animals were manually restrained and abite block placed between the jaws. A pill gun containing the dosingcapsule was inserted into the back of the oral cavity and the capsulereleased. Up to 4 capsules were administered consecutively and a smallamount of water given to induce swallowing. Once the administration ofthe dose was confirmed, the bite block was removed and the headreleased.

Blood samples were collected in K₂EDTA tubes from each animal pre-doseand at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 24 hours post-dose. Animals werereturned to home cages following the 2-hour sample collection. Followingphysical restraint, blood samples were collected via direct venouspuncture using a cephalic, femoral or saphenous vein, which wereprocessed to plasma and stored frozen at −60° C. until shipment.

Results

The concentration-time profiles for total IL-10 in plasma for all dosinggroups are presented in FIG. 58. Following oral administration of theIL-10 delivery construct by capsule at 82 mg (4×20.5 mg capsules),plasma concentration of total IL-10 reached a Cmax of 1.34 μg/mL at aTmax of 2 hours post dose. Dosing with 20.5 mg (1×20.5 mg capsule) IL-10delivery construct also resulted in a Tmax of 2 hours post dose, with asignificantly lower Cmax (0.45 μg/mL) than the 82 mg dose. The Tmax forthe 1 mg (1×1 mg capsule) and 4 mg (4×1 mg capsules) doses was laterthan the higher doses (4 hours and 3 hours respectively), and notably,the Cmax for the 1 mg dose was higher than that of the 4 mg dose (0.39vs. 0.27 μg/mL respectively). At 6 hours post-dose, the plasmaconcentration of total IL-10 had returned to a baseline level.

The concentration-time profiles of IL-1Ra in plasma for all dosinggroups are presented in FIG. 59. After oral dosing with 82 mg IL-10delivery construct, NHP IL-1Ra plasma concentration reached a Cmax of26,367 μg/mL at 3 hours post-dose. No dose response relationship wasobserved for Cmax across the lower dose levels (1, 4 and 20.5 mg). Forall dose groups, the plasma concentration of IL-1Ra returned to baselinelevels by the 24-hour post-dose timepoint.

The plasma concentration of caffeine in NHPs following a single dosewith IL-10 delivery construct/caffeine capsules as specified in TABLE 45is presented in FIG. 60. It should be noted that animals in the 1 mg(1×1 mg capsule) and 20.5 mg (1×20.5 mg capsule) dosing groups eachreceived a 1×10 mg dose of caffeine, while animals administered the 4 mg(4×1 mg capsules) and 82 mg (4×20.5 mg capsules) doses each received a4×10 mg dose of caffeine. This explains the significantly higher C_(max)of caffeine seen for the 4 and 82 mg dosing groups (6117 ng/mL and 3793ng/mL respectively) than for the 1 and 20.5 mg dosing groups (1352 ng/mLand 800 ng/mL respectively). Absorption of caffeine was observable 2-3hours post dose (demonstrating opening of capsules) and the Tmax for alldoses was 4 hours post dose.

Administration of IL-10 delivery construct (SEQ ID NO: 5) capsules wasnot associated with any consistent trends in plasma IFNγ (FIG. 61A),IL-10 (FIG. 61B), IL-2 (FIG. 61C), IL-8 (FIG. 61D), or IL-6 levels (FIG.61E), although high variability and marked influence of outliers on themean values for individual timepoints were observed for plasma IFNγ,IL-2, IL-6 and IL-8. Similar quantitation of TNFα showed no induction assignals for all samples were below the limit of detection (data notshown).

Additionally, plasma levels of IL-10, IL-1Ra, and IFN-γ were assessedfollowing administration of IL-10 delivery construct (SEQ ID NO: 5)capsules was compared with intravenous administration of the IL-10delivery construct (SEQ ID NO:5) (FIGS. 104A-104C). Oral delivery of theIL-10 delivery construct (SEQ ID NO:5) in non-human primates viaenteric-coated capsules increased IL-1Ra without significant inductionof IFN-γ.

Discussion and Conclusions

The systemic concentration of the IL-10 delivery construct (as measuredby total IL-10) increased significantly above baseline levels at alldose levels (1, 4, 20.5 and 82 mg), and returned to baseline levelswithin six hours of dose administration. There appeared to be no cleardose relationship for total IL-10 exposure, although the highest dose ofthe IL-10 delivery construct (82 mg) resulted in the highest C_(max)IL-1Ra was induced by IL-10 delivery construct administration in alldosing groups with no clear dose relationship although, as for totalIL-10, the highest dose of the IL-10 delivery construct (82 mg) resultedin the highest C_(max).

Plasma caffeine concentration was used as a marker for capsule openingand performance. Absorption of caffeine was observable 2-3 hours postdose (demonstrating opening of capsules) and the Tmax for all doses was4 hours post dose.

Administration of the IL-10 delivery construct capsules was notassociated with any consistent trends in plasma IFNγ, IL-1β, IL-2, IL-6or IL-8 levels.

Example 17—Cell Targeting in the Lamina Propria

Male adult (7-8 week old) Wistar rats with an average weight of about250 g were used to perform in vivo studies examining cell targeting inthe lamina propria of an IL-10 delivery construct (SEQ ID NO: 5). Ratswere anesthetized using inhaled isoflurane and euthanized by inhaledCO₂.

Experiments were initiated by making an approximately 4 cm abdominalincision to access the mid-jejunum region of the small intestine. Afterperforming the incision, 50 μl of a prepared solution of the IL-10delivery construct (typically at about 1 mg/mL) was injected into theintestinal lumen of an area devoid of foodstuffs through a 27-gaugeneedle using a 1 mL syringe. The mesentery adjacent to the site ofinjection was labeled with a marker and the intestine was returned tothe abdominal cavity, with the incision being closed with clamps.

At specific times, the injected intestine was retrieved, surgicallyisolated and flushed with a 4° C. isotonic PBS. Washed, excised sampleswere fixed (4% paraformaldehyde in PBS) overnight at 4° C. beforedehydration through graded series of ethanol/water solutions andovernight incubation in chloroform.

Dehydrated tissues were immersed in wax, sectioned, and mounted onpoylysine slides and processed for antigen retrieval using sodiumcitrate. Afterward, sections were permeabilised with 0.2% Triton-X100 inPBS prior to thrice washing in PBS and blocking with 2% BSA+2% serum ofthe animal the secondary antibodies have been raised. Primary antibodieswere diluted in 1% BSA, 0.1% Triton-X100 in PBS and incubated overnightat 4° C. in humidified air. Fluorescent secondary antibodies werediluted in 1% BSA, 0.1% Triton-X100 in PBS and incubated for 2 h at RTprior to processing for confocal microscopy. On occasion, anapproximately 1 cm section of intestine at the injection site wascollected for biochemical studies.

Proteins for specific cell types were examined by immuno-fluorescenceusing confocal microscopy. The IL-10 delivery construct components (thecarrier, also referred to herein as “cholix,” and IL-10) were followedseparately with either a polyclonal antibody (pAb) or monoclonalantibody (mAb) to IL-10 or a pAb or mAb to the carrier domain to allowco-localization with pAb or mAbs available (TABLE 46).

TABLE 46 Summary of pAbs and mAbs Dilution pAb/ Species for Cells TargetmAb reactivity Host IHC (P) labeled Cat. # Carrier pAb Rabbit 1/500IL-10 pAb Human Goat 1/25  Ab10775  CD11c Dendritic cells CD19 mAb MouseMouse B cells Ab25177  CD34 mAb Mouse, Mouse 1/500 Endothelia Ab187282Human CD3 mAb Rat Mouse T cells Ab185763

The IL-10 delivery construct showed little or no co-localization withLAMP1-positive lysosomes in enterocytes, but did collect within cells ofthe lamina propria there consistent with their ultimate consumptionwhere this protein was directed to lysosomes (FIG. 62). A 1-minute pulsewas performed, followed by a time course study that verified by 15 minan extensive amount of the IL-10 delivery construct reached the laminapropria and entered into cells targeted by the carrier component of thisconstruct.

CD11c antigen labeled a sparse set of cells (e.g. dendritic cells)within the lamina propria of the rat jejunum. The IL-10 deliveryconstruct did not co-localize with CD11c antigen to any striking extent(FIG. 63). Interestingly, vesicles near the apical surface ofenterocytes co-labeled for CD11c and IL-10, suggesting an interactionbetween the carrier protein and a vesicular compartment that harboredthis antigen.

The IL-10 delivery construct failed to co-localize with the fewCD19-positive cells (e.g. B-lymphocytes) within the lamina propria (FIG.64).

The IL-10 delivery construct failed to co-localize with the fewCD34-positive cells (e.g. endothelia) within the lamina propria (FIG.65).

The IL-10 delivery construct demonstrated striking co-localizations withthe CD3 antigen, suggesting significant targeting of the carrier proteinto T lymphocytes within the lamina propria (FIG. 66). Additionally,cells consistent with intra-epithelial lymphocytes were also a site ofco-localization.

Cells within the lamina propria of mouse jejunum showed some of the sametargeting outcomes as that observed for rat. The complication of thesestudies, however, is the ability of human IL-10 to be recognized by themouse IL-10 receptor, leading to two potential cellular fates in somecases.

In conclusion, the carrier in the IL-10 delivery construct used tofacilitate the transcytosis of human IL-10 across enterocytes appears toaccess a select population of cells within the lamina propria. The fateof the carrier within these targeted cells appears to intersect with aLAMP1-positive compartment that is most likely the lysosome, suggestingits apparent local destruction within the lamina propria. T-lymphocytesappear to be the largest cell type present within the lamina propriathat were targeted by the carrier used in the IL-10 delivery construct.

Example 18—Comparison of Tablets Vs. Capsules

To evaluate the drug release from IL-10 delivery construct (SEQ ID NO:5) capsules, and to compare the performance to those of tablets, IL-10delivery construct (SEQ ID NO: 5) capsules were obtained with variouscoating conditions, from HPMC coating only to Eudragit coating atdifferent weight gain.

Capsules with only HPMC coating showed significantly higher IL-10delivery construct (SEQ ID NO: 5) dimer purity and concentrationcompared to those with enteric coating. While, as expected, the presenceof Eudragit coat caused delayed release of IL-10 delivery construct (SEQID NO: 5), the impact on dimer purity and quantity of active drugreleased contrasts with the results obtained for HPMC coated tabletsunder the same dissolution conditions (FIG. 68). Dissolution of size 1HPMC capsules containing 20 mg of the IL-10 delivery construct (SEQ IDNO: 5) and coated with varying amounts (from about 10 mg to about 35 mg)of 50:50 coat weight ratio of Eudragit® L30D55: Eudragit® FS30D wasexamined. The Eudragit coating delayed release of the IL-10 deliveryconstruct from the capsule but had a negative effect on IL-10 deliveryconstruct dimer purity (dimer %) (FIG. 68). Dissolution was measured ona Type 4 dissolution apparatus, flow-through mode, in pH 7.0 dissolutionbuffer at 37° C. The in vitro dissolution test which is used for thesedeterminations is one of several that are standard in the art,particularly for extended release tablets e.g. see USP <711> Dissolutionor Ph. Eur. 2.9.3. Testing for the IL-10 delivery construct is conductedin USP Apparatus 4 (a flow-through cell apparatus, open mode, in pH 7.0dissolution buffer at 37° C.). Individual tablets are placed in theapparatus and sampling is conducted at the specified times (e.g. 10, 20,30 and 60 min). The sampling was conducted by collecting flow throughsolution from the tubing leading to waste bottle. The sample solution inwas kept in vials on ice before SEC HPLC analysis. At each timepoint,the concentration of the IL-10 delivery construct in the fluid sample isdetermined (e.g. by a validated HPLC method), thereby permittingcalculation of the amount which has been released from a tablet.

In contrast, for tablets, the Eudragit coating delayed release of theIL-10 delivery construct from the tablet without impacting the IL-10delivery construct dimer purity (dimer %) (FIG. 69). Tablets were of theF3 tablet formulation, with an IL-10 delivery construct strength of 6mg, spray coated with HPMC, and then had a coat weight ratio of 50:50 ofEudragit® L30D55: Eudragit® FS30D. Two different Eudragit coat weightswere examined (8 mg and 20 mg). Dissolution was measured on a Type 4dissolution apparatus, flow-through mode, in pH 7.0 dissolution bufferat 37° C.

The quantity of IL-10 delivery construct (SEQ ID NO: 5) released wascalculated for tablets and capsules with different amounts of coating(FIG. 70). Related impurities (HMW aggregates, and LMW monomer) werequantified by SEC. Summation of the active (dimer) and impuritiesprovided the total quantity of IL-10 delivery construct (SEQ ID NO: 5)related materials released from the formulation. The results obtainedfurther indicated the performance difference between tablets andcapsules with respect to IL-10 delivery construct (SEQ ID NO: 5)release.

In general, coated tablets showed comparable release of IL-10 deliveryconstruct (SEQ ID NO: 5) dimer to non-coated tablets. The total amountof IL-10 delivery construct (SEQ ID NO: 5) related substances releasedfrom the tablets was generally close to the amount of IL-10 deliveryconstruct in the tablet, although some loss of protein was observed athigher coating levels. In addition, the relative dimer purity of IL-10delivery construct (SEQ ID NO: 5) dimer when considered for all materialover the course of dissolution study was approximately 50% of the totalreleased material for all tablets. Eudragit-coated capsules showed arelatively low proportion and quantity of IL-10 delivery construct (SEQID NO: 5) dimer release, with the majority of the protein identified asHMW aggregates. The total protein released was also reduced, presumablyas a result of insoluble species formed on advanced aggregation.

The extent of inactive components such as high molecular aggregates andmonomer can also be determined in this model. In tablet dissolutiongroup shown at left side of FIG. 70, non-coated tablet was used asreference, and the coating composition varied from 50:50 (FS to L30D) to80:20 (FS to L30D) at increased coating weight gain of 8 mg, 13 mg and20 mg.

The improved performance of tablets relative to capsules may result fromthe form of the API within each formulation, the nature of thedissolution process, and/or the relative amount of Eudragit.

The coating weight on enterically-coated tablets can be significantlyless than on capsules, because of the lower surface area for a given APIweight (compressed tablet vs powder fill to capsule), and also thetablet surface geometry, without the joint between capsule cap and body,making it easier to achieve a uniform tablet coating for a given weight.Tablets may use a lower coat thickness. Thus, due to a smaller surfacearea and/or smaller thickness, the tablets may have less polymer (e.g,Eudragit) per amount of IL-10 delivery construct relative to a capsule.

Additionally, the nature of capsule dissolution may be detrimental toIL-10 delivery construct (SEQ ID NO: 5) release. Encapsulated IL-10delivery construct may be disposed relatively loosely within in a powderfrom within the capsule body. Upon initial access of fluid through thedissolving capsule, a substantially (e.g., the entire) quantity ofprotein may be wetted. Such rapid wetting may lead to aggregation orotherwise disrupt dimer structure. In contrast, in a tablet, the IL-10delivery construct may be blended and compressed in an insoluble matrixwhich is not immediately wetted throughout on initial fluid contact, butdisintegrates and dissolves over a longer period of as the capsuledissolves. This may reduce the propensity towards aggregation duringtablet dissolution and increase the likelihood of maintaining IL-10delivery construct (SEQ ID NO: 5) in the active dimer form.

Example 19—Comparison of Coated Tablets Vs. Uncoated Tablets in a Type 4Dissolution Device

It was hypothesized that the lower IL-10 delivery construct (SEQ ID NO:5) dimer purity observed on dissolution of coated tablets relative touncoated tablets may reflect the simultaneous presence in solution ofdissolved Eudragit polymers and IL-10 delivery construct (SEQ ID NO: 5),since this combination was previously shown to cause loss of IL-10delivery construct (SEQ ID NO: 5) dimer. However, this may not reflectthe in vivo situation for tablet dissolution, as the coating materialmay have more limited contact with IL-10 delivery construct (SEQ ID NO:5) in the GI tract following oral administration. Instead, as the tabletmoves along the gut, coating material could be gradually removed fromtablet core prior to the complete release of the drug, thus separatingthe coating materials from the protein.

An in vitro flow through method with USP dissolution type-4 apparatus(FIG. 98) was implemented to better predict drug release in the GItract. In comparison to the type-2 apparatus (dissolution conducted in astirred chamber with no active flow) the type-4 instrument can operatein “open mode”, whereby a constant influx of dissolution medium isdelivered to maintain infinite sink conditions (i.e., no local buildupof protein or tablet components).

Coated and uncoated IL-10 delivery construct (SEQ ID NO: 5) tablets wereevaluated by in vitro dissolution using the type-4 apparatus in openmode (flow-through) (FIG. 69). As expected, release of IL-10 deliveryconstruct (SEQ ID NO: 5) was delayed as a function of increasing coatingweight, but the release profile (dimer content) was not affected bycoating, showing comparable initial dimer purity and degradation ratefor each tablet (FIG. 69).

Coated tablets also showed delayed release, but IL-10 delivery construct(SEQ ID NO: 5) dimer content was substantially reduced compared touncoated tablets, presumably due to the incompatibility of IL-10delivery construct (SEQ ID NO: 5) and Eudragit polymers. It is expectedthat the type-4 instrument provides an environment which is morerepresentative of the behavior of a coated IL-10 delivery construct (SEQID NO: 5) tablet in the GI tract and thus should better simulate in vivoperformance.

As well as monitoring IL-10 delivery construct (SEQ ID NO: 5) dimerpurity, the quantity of drug released at each time point was obtainedduring type-4 dissolution (FIG. 98). Each tablet showed a similarduration of drug release, with increasing coating amounts delaying drugdissolution. These data suggested that once tablet disintegrationstarted, the release process progressed in a similar manner, with thecoated tablet core undergoing the same surface hydration anddisintegration process once the coating material was removed, regardlessof coating thickness. Thus the delayed release feature provided byenteric coating showed little if any impact to the drug quality in thetablet core.

A second set of tablets coated with a different Eudragit formulationshowed similar dissolution behavior (FIG. 99), further supporting thatrelease of IL-10 delivery construct (SEQ ID NO: 5) was not significantlyaffected by the coating under these conditions in the Type 4 apparatus.

Example 20—HPMC-AS Coating of Tablets

Alternatives to Eudragit coatings were also evaluated. AQOAT®(Shin-Etsu) hypromellose acetate succinate (HPMCAS) was selected for itssimilar pH responsive properties, and for its weak ionic structurecharacteristic. These characteristics are in contrast to the strongionic structure of Eudragits and could lead to better compatibility toIL-10 delivery construct (SEQ ID NO: 5) structural integrity during thedrug tablet coating and drug release processes. A preliminary screeningof compatibility was conducted. Eudragit L30D-55 and FS 30D werecompared to AQOAT® products AS-HF and AS-MF, which reportedly becomesoluble at pH 6.8 and pH 6.0, respectively. The dimer purity of theIL-10 delivery construct (SEQ ID NO:5) incubated with various coatingagents in PBS, as measured by dimer content, is shown in TABLE 47.Incubation with AS-HF and AS-MF showed little or no degradation of theIL-10 delivery construct (SEQ ID NO: 5), indicating significantimprovement of drug compatibility by such HPMC-AS derivatives.

TABLE 47 Compatibility of coating materials with IL-10 deliveryconstruct (SEQ ID NO: 5); 2 h incubation with IL-10 delivery construct(SEQ ID NO: 5) protein in PBS, structure integrity was analyzed by SECmethod Component Dimer Monomer HMW IL-10 delivery construct 88.1 8.8 3.2(SEQ ID NO: 5) Eudragit FS30D 45.8 12.2 42.0 Eudragit L30D55 12.6 45.242.1 HPMCAS-HF 86.2 11.5 2.3 HPMCAS-MF 88.3 8.7 3.0

IL-10 delivery construct (SEQ ID NO: 5) tablets coated with 50:50 AS-HFand AS-MF were evaluated by dissolution on type-4 device (FIG. 100, FIG.101). The initial dimer purity of released material was found to beessentially identical to the drug substance (lyophilized IL10 deliveryconstruct), indicating that the HPMCAS coating has little impact on thedimer purity of the IL-10 delivery construct (SEQ ID NO: 5). The delayof release correlated with tablet coating thickness, showing comparablerelease rate after initial delay, and similar to that of non-coatedtablets, further indicated no negative effect of the coating materialsduring tablet disintegration.

HPMC-AS coated tablets showed essentially equivalent or improved releaseprofiles when compared to Eudragit-coated IL-10 delivery construct (SEQID NO: 5) tablets, as characterized by drug purity, degradation rate,and drug exposure (AUC of dimer concentration) in vitro. This wasfurther illustrated by dissolution testing on a type-2 apparatus (FIG.102, FIG. 103). Relative to testing in a Type-4 dissolution apparatus,the impact of incompatibility between coating and IL-10 deliveryconstruct became more visible in such a setting. The actual in vivorelease is likely to be more analogous to the conditions employed with aType 4-dissolution apparatus. Nonetheless, in both cases, the HPMC-AScoatings displayed advantageous drug stability profiles over Eudragitcoatings.

The dimer purity and concentration of IL-10 delivery construct (SEQ IDNO: 5) release were plotted against the dissolution time. On a type-2dissolution apparatus, tablets with increased coating weight correlatedwith a longer delay of release. The drug concentration reached tomaximum cumulative concentration in 20-30 minutes after initialdisintegration, correlating to what was observed in dissolution ontype-4 device that the disintegration completed in a similar timeinterval after the coating was initially breached.

In summary, HPMCAS coating materials demonstrated superior chemicalcompatibility with IL-10 delivery construct (SEQ ID NO: 5) dimerrelative to Eudragits. HPMCAS-coated IL-10 delivery construct (SEQ IDNO: 5) tablets displayed essentially equivalent or improved releaseprofiles in terms of dimer purity and exposure in two commonly used invitro dissolution models. The two stage dissolution on flow throughmodel confirmed that the coating was pH responsive as it was stable atlower pH and becoming labile at high pH for drug release. The HPMCAScoating provided protection of the IL-10 delivery construct at a low pHwas found effect The drug protection by coating at low pH was foundeffective as the drug exposure in dissolution test remained same with orwithout acid treatment.

Example 21—Assessment of Pharmacokinetics and Pharmacodynamics of anIL-10 Delivery Construct (SEQ ID NO: 5) Through Pan-ColonicAdministration Via Sigmoidoscopy in Non-Human Primates

This non-GLP study was conducted to investigate PK and PD in malecynomolgus monkeys (n=3/group) administered a single dose of 1, 3 or 10mg IL-10 delivery construct (SEQ ID NO: 5) through pan-colonicadministration via sigmoidoscopy. IL-10 delivery construct solution (atconcentrations of 0.1, 0.3 and 1.0 mg/mL for the low, mid and high-dosegroups respectively) was administered via the rectal route usingsigmoidoscopy and a syringe connected to a spray nozzle with a 3600spray pattern. Serial biopsy samples were collected by sigmoidoscopy(pre-dose, 0.25, 0.5, 0.75, 8 and 24-28 hours post dose). Serial plasmasamples from 0 min pre-dose to between 24-28 hours post-dose werecollected for analysis for the IL-10 delivery construct.

Plasma Concentration of Total IL-10 and IL-10 Delivery Construct (SEQ IDNO: 5)

Exposure, determined by total IL-10 concentration, resulting from asingle dose of IL-10 delivery construct (SEQ ID NO: 5) delivereddirectly to the colon via sigmoidoscope was detected in the plasmawithin 15 minutes of administration (FIG. 71A). Exposure to total IL-10increased with dose and t_(max) occurred within one hour of delivery inall three dose groups (1, 3, and 10 mg) (FIG. 71A). In addition to totalIL-10 analysis, IL-10 delivery construct (SEQ ID NO: 5) concentrationwas determined by an anti-cholix³⁸⁶ capture antibody, in conjunctionwith an anti-human IL-10 detection antibody. IL-10 delivery construct(SEQ ID NO: 5) concentration (FIG. 71B) were 10-fold lower than those ofIL-10 at 3 mg and 10 mg doses (FIG. 71A). A dose-dependent response inIL-10 delivery construct (SEQ ID NO: 5) was not revealed between thegroups. The lower plasma IL-10 delivery construct (SEQ ID NO: 5)concentrations, compared to total IL-10, imply that circulating IL-10delivery construct (SEQ ID NO: 5) may be comprised of cleaved orotherwise processed versions of IL-10 delivery construct (SEQ ID NO: 5),in which the cholix³⁸⁶ domain (SEQ ID NO: 4) was no longer conjugated torhIL-10.

Plasma Concentration of IL-1Ra Following IL-10 Delivery Construct (SEQID NO: 5) Administration

An induction of the plasma concentration of IL-1Ra was observed at allthree doses (1, 3 and 10 mg). IL-1Ra was detected at higherconcentrations in response to the 3 and 10 mg doses, compared to 1 mg(FIG. 71C); however high intra-group variability was detected and aclear dose-dependent response was not observed. The absence of adose-dependent effect at the higher concentrations of IL-10 deliveryconstruct (SEQ ID NO: 5) suggested that the IL-10 induction of IL-1Rawas saturated at the 10 mg dose. Total IL-10 peaked at approximately0.5-1 h (FIG. 71A), and maximal plasma IL-1Ra concentration followed at3-4 h (FIG. 71C). Despite considerable variations in exposure and IL-1Raproduction between animals, the relative kinetics of total IL-10 andIL-1Ra detection suggested that IL-1Ra induction may be mediated byIL-10 delivery construct (SEQ ID NO: 5).

Plasma Concentration of Pro-Inflammatory Cytokines Following IL-10Delivery Construct (SEQ ID NO: 5) Administration

Colonic administration of IL-10 delivery construct (SEQ ID NO: 5) didnot lead to an increase in the systemic concentrations ofpro-inflammatory cytokines IFN-γ, IL-10, IL-2 or IL-8, but IL-6 wasmoderately induced in the 3 and 10 mg groups (FIG. 72).

Exposure, assessed by total IL-10 concentration, from a single dose ofIL-10 delivery construct (SEQ ID NO: 5) delivered directly to the colonvia sigmoidoscope was detected in the colonic tissue (FIG. 105C) andsystemic circulation (FIG. 105A). Exposure was also assessed by IL-1Raconcentration in systemic circulation (FIG. 105B). A dose-dependentincrease in total IL-10 was detected in the tissue, which peaked at thefirst sampling time point of 15 minutes. In addition to total IL-10analysis, the concentration of IL-10 delivery construct (SEQ ID NO: 5)in colonic tissue was determined. Tissue IL-10 delivery construct (SEQID NO: 5) concentrations did not reveal clear dose dependence among thegroups (FIG. 105D). IL-10 delivery construct (SEQ ID NO: 5)concentration was increased 15 minutes following the 1 and 10 mg doses,with high variability detected within the groups.

IL-1Ra Concentration in Colonic Tissue Following Pan-Colonic IL-10Delivery Construct (SEQ ID NO: 5) Administration

IL-1Ra concentration was assessed in the colonic tissue of NHP followingpan-colonic delivery at IL-10 delivery construct (SEQ ID NO: 5) at 1, 3,and 10 mg doses. High concentrations of endogenous IL-1Ra were detectedin colon tissue lysates at baseline and IL-1Ra concentration was notfurther augmented in response to the IL-10 delivery construct (FIG. 73).

Phosphorylation of STAT3 in Tissue Following Pan-Colonic IL-10 DeliveryConstruct (SEQ ID NO: 5) Administration

Phosphorylation of STAT3 is an early downstream event following IL-10receptor activation. STAT3 phosphorylation was investigated in colonictissue samples from several time points (15, 30, and 45 minutes, 8hours, and 24 hours) following IL-10 delivery construct (SEQ ID NO: 5)administration. Higher ratios of colonic tissue pSTAT3/STAT3, indicativeof colonic STAT3 activation, were observed following IL-10 deliveryconstruct treatment at all doses tested (FIG. 74).

Analysis of Pro-Inflammatory Cytokine Concentrations in Colonic TissueLysates Following Pan-Colonic Administration of IL-10 Delivery Construct(SEQ ID NO: 5)

Induction of pro-inflammatory cytokines in colonic tissue was notobserved; however, there was evidence for an increase in plasmaconcentrations of IL-6 with IL-10 delivery construct treatment (FIG.75).

Colonic biopsies at each tissue timepoint were analyzed for localconcentration of IL-10 delivery construct, total IL-10, IL-1Ra, solublecytokines, activation of STAT3 and gene expression followingadministration of the IL-10 delivery construct. The IL-10 deliveryconstruct treatment upregulated a number of anti-inflammatory genes,notably including CD163, miR24-2 and NOX1, which play roles in thedifferentiation or function of tolerogenic M2 macrophages. In addition,STC1, MTNR1A and VNN2 encode proteins that suppress the NFkβ signalingpathway, a pathway through which key pro-inflammatory cytokines areregulated. The IL-10 delivery construct treatment downregulated theexpression of the drug metabolizing enzyme CYP1A1; however, the 12-hourfast prior to treatment could have influenced CYP1A1 expression (FIG.76). IL-10 delivery construct administration decreased a number ofpro-inflammatory genes including MHCII and PLA2G2D, which are expressedby dendritic cells and macrophages. This is consistent with the abilityof IL-10 to suppress these cells' activation or differentiation towardsa pro-inflammatory immune phenotype. In addition, the IL-10 deliveryconstruct downregulated CCL20 and CCL13, chemoattractants that recruitmacrophages and innate immune cells, respectively (FIG. 77). With IL-10delivery construct treatment, a number of pro-inflammatory genes werealso upregulated, including matrix metalloproteinases: MMP1, MMP3 andMMP19 (FIG. 78). Importantly, consistent with the regulatory function ofIL-10, the IL-10 delivery construct also increased a matrixmetalloproteinases inhibitor, TIMP1, as well as other tissue repair(FIG. 79) and anti-microbial genes (FIG. 80).

Example 22—Assessment of Effects of an IL-10 Delivery Construct (SEQ IDNO: 5) in a Mouse Model of Ulcerative Colitis

This study was conducted to investigate the efficacy of oral treatmentwith an IL-10 delivery construct (SEQ ID NO: 5) in a murineoxazolone-induced ulcerative colitis model. Oxazolone-induced colitis inmice constitutes an animal model of UC with similarity to thehistopathological characteristics and distribution of inflammationdescribed in human ulcerative colitis and can be used to screenpotential therapeutic agents.

Healthy young female SJL/J mice were pre-sensitized with 200 μl of 3%(w/v) solution of oxazolone(4-ethoxymethylene-2-phenyl-2-oxazoline-5-one) in 100% ethanol, appliedtopically. Five days after pre-sensitization, mice were challengedintra-rectally with 150 μL of 1% oxazolone in 40% ethanol. Control micewere pre-sensitized with 100% ethanol on day −5 followed by intra-rectaladministration of 150 μL of 40% ethanol (no oxazolone). Mice were dosedby oral gavage once a day (q.d.) with the IL-10 delivery construct,5-ASA (100 mg/kg, 15 mice) or Vehicle from day −5 to day +6 for a totalof 12 doses. The IL-10 delivery construct was provided at four differentdoses: 9 mg/kg, 3 mk/kg, 1 mg/kg, and 0.3 mg/kg. There were 15 mice ineach treatment group, 10 mice in the vehicle treated group, and 5 micein the naïve control group.

Blood samples were collected into microtainer tubes (BD 365974)containing EDTA and centrifuged at 500 RCF at room temperature (RT) for10 minutes. Supernatants were collected and transferred into a cleantube and re-centrifuged at 13,000 RCF RT for 10 minutes. Plasma sampleswere aliquoted into two 80-100 μL size aliquots and stored at −80° C.Jejunum and ileum from each surviving mouse were removed and cleared ofall fecal content with cold PBS and approximately 1 cm of each sectionwas flash-frozen. The colon (minus cecum) from each of the survivingmice was removed and cleared of all fecal matter with cold PBS. Colonlength and weight were recorded. Samples were cut from the colon atdifferent points along the length of the colon (region 1 proximal,region 2 mid, region 3 between regions 2 and 4, and region 4 distal) andpreserved in 10% neutral formalin buffer for 24 hrs. and thentransferred to 70% EtOH. These samples were sectioned and stained byhematoxylin and eosin staining or immune fluorescence. Region 3 of thesame colons was flash-frozen.

Treatment with the IL-10 delivery construct (SEQ ID NO: 5) resulted inimproved colonic histopathology. FIG. 81A shows hematoxylin and eosinstaining of a naïve colon, while FIG. 81B shows a colon section from amouse which was treated with oxazolone to induce a disease state, butreceived vehicle in the treatment phase. The colonic histopathology isimproved in FIG. 81C which shows a section from a representativeoxazolone induced mouse treated with an IL-10 delivery construct (8.5mg/kg).

Treatment with an oral IL-10 delivery construct (SEQ ID NO: 5) decreasedexpression of inflammatory markers IL-4 (FIG. 82A), IL-6 (FIG. 82B),IL-10 (FIG. 82C), IL-17A (FIG. 82D), IL-10 (FIG. 82E), MIP1α (FIG. 82F),and GCSF/CSF3 (FIG. 82G). Statistical analysis was performed using a1-way ANOVA with Tukey's post test.

Treatment with the oral IL-10 delivery construct (SEQ ID NO: 5) inducedupregulation of tissue and circulating biomarkers. FIG. 84 shows thatsystemic expression of IL-1Ra increased in a dose dependent manner upontreatment with the IL-10 delivery construct (doses are shown on x axisin mg/kg). Treatment with 9 mg/kg of the IL-10 delivery construct (SEQID NO: 5) increased colon expression of IL-1Ra compared to vehicle (FIG.85A), decreased colon expression of IL-10 compared to vehicle (FIG. 85B)and increased the ratio of IL-1Ra to IL-10 by about 2.5 fold (FIG. 85C).Treatment with the IL-10 delivery construct increased the ratio ofphosphorylated STAT3 (pSTAT3) to total STAT3 by about 2 fold in smallintestine tissue and about 3 fold in colon tissue (FIG. 86).

Example 23—Assessment of Effects of an IL-10 Delivery Construct (SEQ IDNO: 5) on STAT3 in Colon Tissue in Non-Inflamed Mice

To assess the effect of an oral IL-10 delivery construct on STAT3 innon-inflamed colon tissue mice were administered with vehicle, an IL-10delivery construct (1 mg/kg) or recombinant human IL-10 (rhIL-10, 0.9mg/kg, an equimolar dose to that of the IL-10 delivery construct. TotalSTAT3 and phosphorylated STAT3 (pSTAT3) were assayed by ELISA MSD. FIG.83A shows that treatment with the IL-10 delivery construct resulted inan approximately 50% increase in the ratio of pSTAT3 to total STAT3,while the rhIL-10 treatment did not significantly alter the ratio ofpSTAT3 to total STAT3.

To assess the effect of an oral IL-10 delivery construct on IL-1Ra innon-inflamed colon tissue mice were administered with vehicle, an IL-10delivery construct (10 mg/kg) or recombinant human IL-10 (rhIL-10, 3mg/kg, an equimolar dose to that of the IL-10 delivery construct.Systemic IL-1Ra was assayed by ELISA MSD. FIG. 83B shows the level ofsystemic IL-1Ra was increased by about 3-fold in the mice treated withthe IL-10 delivery construct, while the treatment with rhIL-10 did notsignificantly increase systemic IL-1Ra levels.

Example 24—Assessment of Effects of an IL-10 Delivery Construct (SEQ IDNO: 5) on Immune Homeostasis and Wound Healing in Non-Human Primates

To assess the effect of an IL-10 delivery construct on pro-inflammatoryand anti-inflammatory markers the IL-10 delivery construct was deliveredto male Macaca fascicularis monkeys (about 5 to about 8 kg) by colonicsigmoidoscopy with a spray nozzle. The IL-10 delivery construct wasadministered at doses of 1 mg, 3 mg, and 10 mg formulated in albumin.Treatment with the IL-10 delivery construct decreased expression ofpro-inflammatory markers (FIG. 87), increased expression ofanti-inflammatory markers (FIG. 88) and increased expression ofbiomarkers associated with tissue repair and wound healing (FIG. 89).

Example 25—Assessment of the Pharmacokinetics and Pharmacodynamics of anIL-10 Delivery Construct (SEQ ID NO: 5) in Non-Human Primates

To compare different routes of delivery an IL-10 delivery construct (SEQID NO: 5) was administered to Macaca fasciularis monkeys orally at 1 mg(N=6) or 5 mgs (N=6), subcutaneously at 0.2 mg/kg (N=3), orintravenously at 0.05 mg/kg (N=3). For oral administration the IL-10delivery construct was formulated in size 0 enteric coated capsules. Forintravenous or subcutaneous administration it was formulated as a liquidin DS lyo formulation. Oral treatment with the IL-10 delivery constructresulted in minimal systemic levels of IL-10 compared to either thesubcutaneous or intravenous administration (FIG. 90A). Systemic levelsof IL-1Ra are shown in FIG. 90B. Oral treatment with the IL-10 deliveryconstruct increased the IL-1Ra/IL-10 ratio to about 15,000:1 at 1 mg,and greater than 20,000:1 for the 3 mg dose, while minimal effect wasseen from either subcutaneous or intravenous administration (FIG. 91).

Example 26—Evaluation of Surfactants

The effect of surfactants on the dimerization of an IL-10 deliveryconstruct (SEQ ID NO: 5), before and after being subjected to shearstress, was investigated. More particularly, various surfactants wereadded to solutions comprising IL-10 delivery construct (SEQ ID NO:5),and the resulting solutions were tested before being subjected to shearstress, and after being vortexed at 800 rpm continuously for 4 hours.The following surfactants were considered: (1) control (1×PBS, nosurfactant), (2) polysorbate 80 (PS80), 0.1% in 1×PBS, (3) polysorbate20 (PS20), 0.1% in 1×PBS, and poloxamer 188 (F68), 0.3% in 1×PBS.

Following vortexing, the solution was visually inspected. The samplewithout a surfactant was slightly hazy while the samples with thesurfactant were clear after vortexing. The turbidity (340-360 nm) of thesolution was assessed after vortexing. Without a surfactant, absorbanceincreased in the 340-360 nm range (FIG. 92), and an increase inabsorbance was also observed with 0.1% PS20.

SEC-HPLC was carried out on the IL-10 delivery construct formulationsbefore (FIG. 93) and after (FIG. 94) vortexing (TABLE 48). These resultsdid not show significant changes before and after vortexing for allsamples of the IL-10 delivery construct. However, polysorbate 80 showedan increase in HMW aggregates and monomers (of the IL-10 deliveryconstruct) before vortexing, indicating some destabilizing effect on theIL-10 delivery construct. Additionally, polysorbate 20 showed the mostamount of monomer before vortexing, indicating the occurrence of somedissociation of the IL-10 delivery construct. In conclusion, thesurfactant F68 (poloxamer 188) at 0.3% seemed to stabilize the IL-10delivery construct in 1×PBS under shear stress.

TABLE 48 Percent IL-10 delivery construct in monomer, dimer, andaggregate (HMW) forms as assessed by SEC-HPLC before and after vortexingBefore vortex After vortex 0.3% 0.1% 0.1% 0.3% 0.1% 0.1% SEC(%) controlF68 PS80 PS20 control F68 PS80 PS20 HMW 0.57 0.49 3.47 0.9 0.82 0.813.43 0.94 Dimer 93.6 94 87.3 82.5 92.5 93.5 86.6 82.9 Monomer 5.81 5.59.2 16.65 6.73 5.66 10 16.17

Example 27—In Vivo Evaluation of pSTAT3

In vivo studies in mice were performed where the biological activity ofhuman IL-10 following transcytosis was assessed for its ability tostimulate the mouse IL-10 receptor and increase phosphorylation of STAT3(pSTAT3) in cells within the lamina propria. These studies demonstratedthat an IL-10 delivery construct (SEQ ID NO: 5) (and not IL-10 alonewithout the carrier molecule) was shown to selectively transport acrossthe epithelial cells and was capable of increasing STAT3 phosphorylationin cells within the lamina propria after transport across epithelialcells. Additionally, IL-10 delivery construct (SEQ ID NO: 5) exposurewas increased in intestinal tissues and systemic exposure was minimized.

Intraluminal injection of IL-10 delivery construct (SEQ ID NO: 5)increased phosphorylation of STAT3 in cells within the lamina propria.Tissue localization of rhIL-10 and pSTAT3 was detected byimmunofluorescence confocal microscopy about 10 minutes afterintraluminal injection of IL-10 delivery construct (SEQ ID NO: 5) intothe jejunum of Balb/C mice (FIG. 107). An extensive amount of rhIL-10(SEQ ID NO: 5) was trafficked through enterocytes. Additionally, therewere a population of lymphocyte-like cells at the basolateral surface ofenterocytes and within the lamina propria that were pSTAT3 positive.Increases in phosphorylation of pSTAT3 over time are shown in FIG. 108.

Trafficking of IL-10 delivery construct across intestinal epithelium wasvalidated in different murine models, as shown by confocal microscopy(FIG. 109).

Entire intestines of mice were isolated and processed as a “swiss role”and localization of pSTAT3 was detected by immunofluorescence confocalmicroscopy (FIG. 110). IL-10 activity was demonstrated along the lengthof intestine (in all intestinal segments, including colon) after oralgavage via detection of STAT3 phosphorylation along lamina propria,indicative of immunological signaling pathway activation.

Example 28—Intraluminal Injections of SEQ ID NO: 5 in the T-CellTransfer Model of Inflammatory Bowel Disease

This study was conducted to examine the potential for the carrierprotein to transport rhIL-10 from the intestinal lumen into thesubmucosal region in inflamed intestinal tissue. The T lymphocyte (Tcell) transfer model was used as it is a well-established model ofinduced chronic colitis in mice and presents many of the essentialimmunological hallmarks observed in IBD patients. Nine BALB/c mice andtwelve SCID mice, including nine with the colitis-like model ofinflammation, were dosed with 20 μl of test article via intraluminalinjection into the distal ileum and the proximal, middle and distalcolon. 159 pmoles IL-10 delivery construct (SEQ ID NO: 5) wasadministered to healthy BALB/c mice and SCID mice with induced colitis,159 pmoles rhIL-10 was administered to healthy BALB/c mice and SCID micewith induced colitis, and PBS vehicle was administered to healthy BALB/cmice and both healthy and induced colitis SCID mice (n=3/group). In eachgroup, two mice were terminated 10 minutes after intraluminalinjections, and one was terminated 40 minutes after intraluminalinjections. Intestinal tissue and serum were collected. Collected tissuewas fixed, embedded, sectioned and H&E stained for histologicalassessment of inflammatory status. ELISA analysis to measure rhIL 10 wasperformed on lysates prepared from intestinal tissue and serum (FIGS.106A-106D).

Histopathological assessment of tissue confirmed inflammation in all theSCID T cell transfer mice at all levels of the colon.

ELISA measurement of rhIL-10 at the 10-minute exposure time point wassubstantially higher in IL-10 delivery construct (SEQ ID NO: 5) injectedmice compared to the mice injected with rhIL-10 alone (FIGS. 106A-106B).Furthermore, IL-10 delivery construct (SEQ ID NO: 5) transported intoinflamed tissues more efficiently than in non-inflamed tissues. Innormal intestinal tissues, IL-10 delivery construct (SEQ ID NO: 5)uptake was two- to five-fold higher compared to mice injected withrhIL-10 alone. In inflamed tissue, rhIL-10 levels measured after IL-10delivery construct (SEQ ID NO: 5) injection were approximately 10-foldhigher than those measured after rhIL-10 alone (FIG. 106B). Recombinanthuman IL-10 detected following IL-10 delivery construct (SEQ ID NO: 5)administration was detected 40 minutes post exposure time in mosttissues, but appeared to be more stable in healthy compared to inflamedtissue (FIGS. 106C-106D).

IL-10 delivery construct (SEQ ID NO: 5), but not rhIL-10 administration,resulted in moderately high rhIL-10 detected in the serum of healthyanimals, in a time-dependent manner. In inflamed animals, minimalamounts of rhIL-10 were detected in the serum, from both IL-10 deliveryconstruct (SEQ ID NO: 5) and rhIL-10 intraluminal injections, indicatingsome mild non-specific leakage in the inflamed intestinal tissues.

The difference in systemic exposure in healthy vs. diseased mice may beattributed to an intestinal ‘sink effect’, that describes the highernumber of infiltrated immune cells in disease state to which IL-10delivery construct (SEQ ID NO: 5) can target. The numerous infiltratingleukocytes, as observed by histopathology in the intestinal mucosa andsubmucosa, restrict IL-10 delivery construct (SEQ ID NO: 5)/rhIL-10 tothe lamina propria while limiting systemic exposure. IL-10 deliveryconstruct (SEQ ID NO: 5) administration resulted in enhanced intestinaluptake of rhIL-10 in colonic tissues, which is evidence that cholix³⁸⁶facilitates the transcytosis of rhIL-10 through the epithelium.

Example 29—Phase 1a Clinical Study

The IL-10 delivery construct (SEQ ID NO: 5) was designed to assess thesafety and tolerability, pharmacokinetics and pharmacodynamics ofincreasing single oral doses of the IL-10 delivery construct in healthymale subjects (Part A). In part B of the study, early data was gatheredon the safety and tolerability, pharmacokinetics, pharmacodynamics andearly clinical response following multiple ascending doses of the IL-10delivery construct in Ulcerative Colitis (UC) patients (Part B).

The primary objective was to assess the safety and tolerability ofsingle and multiple ascending doses of the IL-10 delivery construct inhealthy adult volunteers and patients with active UC. Other objectivesof the study were to assess: pharmacokinetics (PK) and pharmacodynamics(PD) of the IL-10 delivery construct, to assess the incidence ofanti-drug antibodies against the IL-10 delivery construct, to evaluatepotential PD biomarkers of the IL-10 delivery construct in plasma andtissue (Part B only), to assess the delivery of the IL-10 deliveryconstruct to colonic tissue based on PD response and to assess clinicalactivity of the IL-10 delivery construct after two weeks of treatment inpatients with active UC.

Phase 1a Trial Design

Part A consisted of a single-ascending dose (SAD) escalation in healthymale volunteers. Six cohorts consisting of orally administered singleascending doses were conducted. At each dose level, 6 subjects wereenrolled, randomized 2:4 to receive a single dose of placebo or theIL-10 delivery construct on day 1. The doses selected were 1 mg, 3 mg,10 mg, 30 mg, 60 mg, and 120 mg.

Preliminary Phase 1a Data

The results of Part A confirm that the IL-10 delivery construct is safeand well tolerated. A total of 3 adverse events occurred, with 1 in 12placebo patients and 2 in a total of 24 active the IL-10 deliveryconstruct patients. All events were mild, self-limiting adverse events.Results of the pharmacokinetic analyses from all doses (1-120 mg)confirmed that the IL-10 delivery construct was gut-selective as no drugwas detected in the blood.

Pre-clinical models have identified peripheral IL-1Ra production as amarker of IL-10 engagement to IL-10R in mucosal lymphocytes. Todetermine if the IL-10 delivery construct was actively transportedacross the epithelial cell lining into the lamina propria and able tointeract with lymphocytes, the levels of IL-1Ra induction was assessed.

The results confirmed that the IL-10 delivery construct was able toinduce IL-1Ra expression (FIG. 111). The loss of immunomodulatoryactivity (IL-1Ra induction) at 60 and 120 mg is consistent with IL10biology.

Example 30—Phase 1b Clinical Study

Data was gathered on the safety, tolerability, pharmacokinetics,pharmacodynamics, and initial clinical response following a multipleascending dose (MAD) of the IL-10 delivery construct in adult patientswith active UC over 14 days of treatment. A lyophilized composition ofthe IL-10 delivery construct and excipients (glycine, sucrose, poloxamer188, and potassium phosphate) was added to size 0 HPMC capsules toprepare three different batches with a dose strength of 1 mg, 5 mg, and20 mg. A coating with a weight ratio of 50:50 of Eudragit® L30D55:Eudragit® FS30D was applied with a thickness of 14.8 to 16.0 mg/cm² anda weight of approximately 76 mg to each capsule. Placebo capsules weregenerated using similarly coated empty capsules.

Phase 1b Trial Design

As shown in FIG. 112, the Phase 1b trial consisted of a MAD escalationin adult patients with active UC. A total of four cohorts were dosedwith either 1 mg, 3 mg, 10, mg or 30 mg of the IL-10 delivery construct,randomized 3:1 to receive the IL-10 delivery construct or a placeboadministered once daily for fourteen days. The goal of the trial was toassess the safety of the IL-10 delivery construct and any change in UCdisease activity, through endoscopy, histology, biomarkers, and serumsamples. Stool samples and colonic biopsies were obtained at baselineand day 14 of treatment to assess fecal calprotectin as well ashistology based upon blinded central read of the Geboes scoring system.Fecal calprotectin is an objective marker of clinical response in UCstudies and the Geboes scoring system is a measure of histologicalresponse. The Geboes scoring system of this example used a 0-22 pointhistologic scoring system in which higher scores represent more severedisease.

Preliminary Phase 1b Data has Demonstrated that Oral IL-10 DeliveryConstruct is Well Tolerated

The results of the Phase 1b trial demonstrated that oral IL-10 deliveryconstruct was observed to be well tolerated by patients with UC. TABLE49 shows that a total of 23 TEAEs were observed including three in thefour placebo patients, and 20 in the 12 active IL-10 delivery constructpatients. The adverse events of the Phase 1b trial includednasopharyngitis and adverse events associated with underlying UCsymptoms such as abdominal pain, diarrhea, and nausea. All adverseevents were self-limiting and mild to moderate, with no adverse eventswarranting early discontinuation of treatment. Importantly, unlikesystemically delivered IL-10 in previous studies, no treatment emergentAEs of anemia or thrombocytopenia were observed.

TABLE 49 Treatment-emergent adverse events (TEAEs) observed betweenactive subjects and placebo 1 mg 3 mg 10 mg 30 mg All Active Placebo (n= 3) (n = 3) (n = 3) (n = 3) (n = 12) (n = 4) # of TEASs 2 7 5 6 20 3 (%pts) (67%) (67%) (67%) (67%) (67%) (50%)

Preliminary Results of Clinical Response

Fecal calprotectin (FCP) is a clinical marker of disease activity inpatients with UC. FCP values greater than 150 μg/g correlate with activeinflammation. As can be seen in FIG. 113, dosing of 1 mg and 3 mg IL-10delivery construct led to placebo-adjusted mean reductions of FCP of 44%and 27% after only 14 days of dosing. Previous clinical studies withsystemically deliver IL-10 showed a diminution of activity at higherdoses, which is also observed with the 10 mg and 30 mg doses of theIL-10 delivery construct.

C-Reactive Protein (CRP) is a biomarker of systemic inflammation. At 1and 3 mgs, greater reductions in CRP levels were observed in UC patientswith baseline CRP greater than 5 mg/L when compared to placebo (FIG.114). By design, the IL-10 delivery construct is a GI-selective proteinand was not detected in systemic circulation. However, reduction in CRPlevels suggests that treating UC patients with oral GI-selective IL-10delivery construct resulted in local intestinal as well as systemicimmunomodulatory activity, which helps enable the treatment ofperipheral inflammatory indications.

The Geboes histologic scoring system is a system that incorporatesimmune cell (lymphocyte and neutrophil) infiltration into the laminapropria and epithelium as well as crypt architecture and destruction,and the presence of ulcerations and erosions. When each component isadded, the total score can range from 0 (normal) to 22 (severeinflammation and tissue destruction). To assess activity of the IL-10delivery construct on the GI mucosa, colonic biopsies were obtained atbaseline and then after 14 days of dosing, and Geboes scores wereassessed by a blinded, central read GI pathologist. The IL-10 deliveryconstruct reduced Geboes scores in 60% (6/10) active IL-10 deliveryconstruct patients when compared to 0% (2/10) placebo patients (FIG.115).

FIG. 116 shows pre-dose and post-treatment histological images from a UCpatient in the Phase 1b trial dosed with 10 mg of the IL-10 deliveryconstruct in which the Geboes score improved from a score of 15 to ascore of three using a 22 point scale, with higher scores indicatingmore severe disease activity. The pre-dose image revealed the UC patienthad crypt destruction and an inflammatory cellular infiltrate in theircolon at baseline which are resolved in the post-treatment image after14 days of treatment with the IL-10 delivery construct.

Example 31: Comparison of Purification of an IL-10 Delivery Constructwith and without Sulfitolysis

IL-10 delivery construct (SEQ ID NO: 5) was purified from inclusionbodies using a process including a sulfitolysis step as described inFIG. 2A, or an analogous process described in FIG. 2B but which did notinclude the sulfitolysis and tangential flow filtration steps betweenthe clarification and refolding steps. Refolding in both processes wascarried out at 4° C. The refolding solution used in both processescontained 1 mM reduced glutathione, 0.5 mM oxidized glutathione, 1MArginine-HCl, 250 mM sucrose, 100 mM Tris pH 8.5 at 4° C., and 2 mMEDTA. IL-10 delivery construct (SEQ ID NO: 5) dimer content wascharacterized by SEC-HPLC following the ultrafiltration/diafiltrationstep after refolding but before AEX chromatography using a TSKgel SW30004 μm, 4.6/300 from Tosoh Biosciences (TABLE 50) and furthercharacterized following AEX chromatography by the Capto Q ImpRes® (GE)chromatogram (TABLES 51-52).

TABLE 50 Percentage of IL-10 delivery construct (SEQ ID NO: 5) invarious forms (high molecular weight aggregates, dimers, and monomers)Process type* Aggregate % Dimer % Monomer % Sulfitolysis of SIB 68 17 8(TFF-2 for 20 h at RT) No Sulfitolysis of SIB 36 27 22 (2x UF/DF at 4°C.) No Sulfitolysis of SIB 57 22 11 (2x UF/DF for 66 h RT) *included inparentheses is a description of the TFF step between refolding and AEXchromatography

TABLE 51 IL-10 delivery construct (SEQ ID NO: 5) yield after Capto QChromatography Capto Capto UFDF Q pool Capto Dimer Q Load concentrationvolume Q Purity Process type (mg) (mg/mL) (mL) Yield (%) Sulfitolysis ofSIB 1680 1.85 270 151 74 (97 mL Capto Q) No Sulfitolysis of SIB 16801.68 270 262 84 (97 mL Capto Q)

TABLE 52 IL-10 delivery construct (SEQ ID NO: 5) dimer recovery afterCapto Q Chromatography Dimer Capto Q Capto Q content in total proteinDimer Dimer Capto Q recovery Recovery Purity Process type Load (%) (%)(%) (%) Sulfitolysis of SIB 20 17 55   82-83 (average of 3) Sulfitolysisof SIB 16.5 9 54.5 74 (97 mL Capto Q) No Sulfitolysis of SIB 25 15.662.4 84 (97 mL Capto Q)

Example 32: Comparison of Purification of an IL-10 Delivery Constructwith and without Sulfitolysis

This example demonstrates the in vivo transcytosis of the deliveryconstruct consisting of the amino acid sequence set forth in SEQ ID NO:5 across intact polarized gut epithelial cells in Wistar rats. The datademonstrates that the delivery construct rapidly and efficientlytransported the IL-10 payload across the polarized gut epithelial cellsinto the lamina propria.

In vivo transcytosis was tested using male Wistar rats. Male Wistar ratswere housed 3-5 per cage with a 12/12 h light/dark cycle and were about225-275 g (approximately 6-8 weeks old) when placed on study.Experiments were conducted during the light phase using a non-recoveryprotocol that uses continuous isoflurane anesthesia. A 4-5 cm midlineabdominal incision that exposed mid-jejunum regions was conducted. Stocksolutions at 3.86×10⁻⁵ M of delivery construct were prepared inphosphate buffered saline (PBS), with 50 μL (per 250 g rat) beingadministered by intraluminal injection (ILI) using a 29-gauge needle.The injection site mesentery was then marked with a permanent marker. Atstudy termination, a 3-5 mm region that captured the marked intestinesegment was isolated and processed for microscopic assessment. The invivo experiments were performed in accordance with the U.K. Animals(Scientific Procedures) Act of 1986, the European Communities CouncilDirective of 1986 (86/609/EEC).

The results of the transcytosis activity of the delivery construct withSEQ ID NO: 5 are shown in FIGS. 117A-117C. The data shows microscopyimages demonstrating transport of the IL-10 payload across polarized gutepithelial cells in Wistar rats at various time points following luminalapplication of the delivery construct with the sequence set forth in SEQID NO: 5 to rat jejunum. The delivery construct (SEQ ID NO: 5) includedthe cholix carrier with SEQ ID NO: 4 (and further including anN-terminal methionine) coupled to the IL-10 payload having an amino acidset forth in SEQ ID NO: 2 via a spacer having an amino acid set forth inSEQ ID NO: 6. Green fluorescence indicates the presence of IL-10 (viastaining with an anti-IL-10 antibody). Blue fluorescence indicates DAPIstaining, which labels DNA, and red fluorescence indicates the presenceof CK-8 (cytokeratin-8) with which a cholix-derived carrier canco-localize (e.g., in a supranuclear region of an epithelial cell)during transcytosis. The white arrows #1 highlight the apical membraneof the epithelial cells, and the white arrows #2 highlight the basalmembrane of the epithelial cells.

FIG. 117A demonstrates the extent of transcytosis of IL-10 one minuteafter luminal application of the delivery construct with the sequenceset forth in SEQ ID NO: 5 to rat jejunum. The data shows that transportof the IL-10 payload from the apical to the basal site and into thelamina propria occurred as early as 1 minute after application of thedelivery construct. White arrow #3 indicates the presence of IL-10 inthe lamina propria.

FIG. 117B demonstrates the extent of transcytosis of IL-10 five minutesafter luminal application of the delivery construct with the sequenceset forth in SEQ ID NO: 5 to rat jejunum. The data shows an increasedamount of transported IL-10 payload that was present in the laminapropria (see e.g., white arrows #3) 5 minutes after luminal applicationof the delivery construct.

FIG. 117C demonstrates the extent of transcytosis of IL-10 ten minutesafter luminal application of the delivery construct with the sequenceset forth in SEQ ID NO: 5 to rat jejunum. The data shows an even higheramount of transported IL-10 payload that was present in the laminapropria (see e.g., white arrows #3) 10 minutes after luminal applicationof the delivery construct.

This data demonstrates that the cholix-derived carrier with the sequenceset forth in SEQ ID NO: 4 (and further including an N-terminalmethionine) is capable of rapidly and efficiently transporting the IL-10payload (SEQ ID NO: 2) across intact polarized gut epithelial cells invivo as demonstrated by the presence of the IL-10 payload in the laminapropria as early as 1 minute after luminal application of the deliveryconstruct (SEQ ID NO: 5). Over the course of this experiment, anincreasing amount of transported IL-10 was detected in the laminapropria.

Example 33: The Elements of the Delivery Construct of SEQ ID NO: 5 areFunctionally Active In Vitro

This example provides a functional assessment of the ability of thecholix-derived carrier (SEQ ID NO: 4) to transport the IL-10 payload(SEQ ID NO: 2) across the intestinal epithelium was verified usingpolarized, confluent monolayers of human small intestinal cells culturedon semi-permeable membranes in vitro (FIG. 118). Despite the much largermolecular mass, the delivery construct (SEQ ID NO: 5) transported acrossthese monolayers to a greater extent than recombinant, human IL-10(rhIL-10), demonstrating that the cholix derived carrier of SEQ ID NO: 5retains its capacity to undergo apical to basal (A→B) transcytosis. Thedelivery construct (SEQ ID NO: 5) was next tested in vitro to ensurethat the IL-10 element of the fusion protein was biologically active. AU2OS osteosarcoma cell line engineered to express the two receptorsinvolved in IL-10 signal transduction (IL-10RA and IL-10RB) and exhibita luminescence event as a result of ligand-induced receptor dimerizationwas used to test increasing doses of the delivery construct, the cholixderived carrier alone and the rhIL-10 alone. The delivery construct hadan EC50 of 971.4 pM compared to 91.53 pM for rhIL-10 (FIG. 119), whilethe cholix derived carrier alone did not induce IL-10R dimerization. Toassess downstream potency, the potential for the delivery construct,rhIL-10, or the cholix derived carrier to induce phosphorylation ofsignal transduction and activator of transcription factor 3 (STAT3) wasexamined using the mouse macrophage J774.2 cells. The delivery constructof SEQ ID NO: 5 demonstrated an EC50 of 263.6 pM compared to 40.98 pMfor a commercial preparation of hIL-10 in this assay (FIG. 120).Notably, the delivery construct has a reduced amplitude of pSTAT3/totalSTAT3 of −1.47% compared to hIL-10 at 2.19%. Finally, pre-treatment ofhuman peripheral blood mononuclear cells (PBMCs) with the deliveryconstruct of SEQ ID NO: 5 resulted in a dose-dependent reduction ofsecreted tumor necrosis factor alpha (TNFα, FIG. 121) and surfaceexpression of HLA-DR (FIG. 122), but not secreted IL-6 (FIG. 123),normally induced by endotoxin (lipopolysaccharide) treatment. Together,these data show that conjoining hIL-10 to the cholix derived carrier ofSEQ ID NO: 4 through a GS polypeptide sequence spacer produced a chimerathat retained the properties required to reach cells within theintestinal lamina propria and to activate IL-10 receptor signalingpathways in that compartment.

Example 34: The Delivery Construct of SEQ ID NO: 5 Transcytoses In Vivo

This example provides an assessment of whether the delivery construct ofSEQ ID NO: 5 can undergo efficient transcytosis in vivo by examiningpSTAT3 induction using of rat intestinal tissue isolated 40 min postoral gavage with PBS, rhIL-10, or the delivery construct.Immunofluorescence microscopy of tissues from PBS-treated animals failedto exhibit the presence of any immuno-reactivity for hIL-10 or pSTAT3induction in either the lamina propria or intestinal epithelium (FIG.124). In some instances, rhIL-10 treated animals exhibited pSTAT3induction in enterocytes but not in cells within the lamina propria; nohIL-10 within the tissue was detected (FIG. 125). This observation isconsistent with findings in mice that intestinal epithelial cells canexpress IL-10 receptors at their apical surface under certain conditionsassociated with development and maintenance of barrier function. Tissuesisolated from animals treated with the IL-10 delivery construct of SEQID NO: 5 demonstrated extensive amounts of hIL-10 and a large number ofcells that were positive for pSTAT3 within the lamina propria (FIG.126). In order to quantify how test articles affected intestinal IL-10signal transduction pathways, image analysis was performed:high-throughput image batches were collected, segmented by epithelialversus lamina propria localization, and then by cellular features.Resulting segmentation patterns allowed binary maps to be created thatwere applied to component data, resulting in intestinal villidistribution scoring of nuclear pSTAT3+ cells (FIG. 127). Tissuestreated with the delivery construct of SEQ ID NO: 5 showed pSTAT3induction in cells both within the lamina propria and in enterocytesthat was greater than that observed in intestinal tissues collected fromPBS- or rhIL-10-treated animals.

This example also assessed whether the delivery construct of SEQ ID NO:5 could transport across inflamed intestinal tissue. A mouse model ofcolitis induced by transfer of CD4+ CD45RBhigh T cells was used in anILI format where ˜160 pmoles of an IL-10 delivery construct or rhIL-10was administered into the distal ileum or proximal, mid, or distalregions of the colon in PBS (FIG. 128). Histopathological microscopicassessment of intestinal tissues confirmed inflammation at all levels ofthe colon but not in the ileum. At 10 min post ILI, distal ileum tissuescontained ˜5-fold higher levels of hIL-10 for the delivery construct ofSEQ ID NO: 5 compared to rhIL-10 treatment; in inflamed tissue hIL-10levels measured after ILI of the delivery construct of SEQ ID NO: 5 were˜10-fold higher compared to rhIL-10 treatment (FIG. 128). The highertissue levels of hIL-10 observed in the inflamed colon relative tonon-inflamed distal ileum was likely due to differences in barrierproperties of these tissues. Tissues examined at 40 min post ILIsuggested that the hIL-10 delivered via the delivery construct of SEQ IDNO: 5 mirrored the data obtained at 10 min with a greater relativeuptake being observed in the non-inflamed distal ileum (data not shown).Importantly, hIL-10 was not observed in the systemic circulation (FIG.128).

Assays were conducted to ensure that the genetic construction of thedelivery construct of SEQ ID NO: 5 retained the A-B transcytosisproperties that should be imparted by the cholix derived carrier thatinvolved receptor-mediated uptake and vesicular trafficking withouttargeting to the lysosomal degradation pathway in polarized enterocytes.The cholix derived carrier and hIL-10 elements of the fusion protein ofthe delivery construct of SEQ ID NO: 5 remained together during A-Btranscytosis and within the lamina propria, allowing localization ofhIL-10 to accurately describe the distribution of the delivery constructof SEQ ID NO: 5 (FIG. 129). While Rab7+ vesicles (defining lateendosomes) were present in both apical and basal vesicular compartmentsof enterocytes, co-localization with the delivery construct occurredpredominantly in the apical vesicular compartment (FIG. 130). Rab11+vesicles defining recycling endosomes) were also observed in both theapical and basal compartments of enterocytes but, opposite to Rab7,co-localizations occurred mostly in the basal vesicular compartment(FIG. 131). The delivery construct of SEQ ID NO: 5 co-localized withRab11 to a greater extent compared to Rab7 within cells in the laminapropria (FIGS. 130 and 131).

MAN1 (Lectin, Mannose Binding 1) protein, which is a resident of theendoplasmic reticulum-golgi intermediate compartment (ERGIC) and knownto be involved in the sorting or recycling of proteins and lipids, hasbeen shown to be involved in cholix transcytosis. The delivery constructof SEQ ID NO: 5 also co-localized with redistributing LMAN1 during A-Btranscytosis (FIG. 132): at 1 min, when the delivery construct was justentering at the luminal membrane, LMAN1 was localized in the apicalcompartment; at 5 min, LMAN1 and some of the internalized the deliveryconstruct of SEQ ID NO: 5 were present in the supranuclear regionassociated with ERGIC distribution; by 10 min the delivery construct ofSEQ ID NO: 5 and LMAN1 were extensively co-localized in the basalcompartment; and by 15 min the extent of LMAN1 redistribution andco-localization with the delivery construct of SEQ ID NO: 5 throughoutthe enterocyte was maximized. The timing of LMAN1 redistribution to thebasal compartment coincides with the presence of hIL-10 being ultimatelydetected in non-polarized cells within the lamina propria where it wasnot associated with LMAN1. This re-distribution of LMAN1 associated withthe delivery construct of SEQ ID NO: 5 A-B transcytosis is identical tothat previously observed for cholix.

The A-B transcytosis pathway accessed by cholix has been shown tointersect with Rab7+ and LAMP1+ vesicles; while these are markers ofboth late endosomes and lysosomes, cholix does not appear to traffic tolysosome-like structures within enterocytes. To examine this point forthe delivery construct of SEQ ID NO: 5, a time course of LAMP1 and thedelivery construct co-localization was performed (FIG. 133). LimitedLAMP1/delivery construct co-localizations were observed in polarizedenterocytes at 1 min post ILI, with the extent of these not increasingover the 15 min time course required for completion of the deliveryconstruct's A-B transcytosis. LAMP1+ structures in non-polarized cellswithin the lamina propria did not show co-localization with the deliveryconstruct of SEQ ID NO: 5 at 1 min and 5 min post ILI. There were,however, extensive LAMP1/delivery construct co-localizations innon-polarized cells within the lamina propria at 10 min and 15 min postILI. Together, these data suggest that the delivery construct of SEQ IDNO: 5's A-B transcytosis completed over a time course of 10-15 min andinvolved apical compartment Rab7+ vesicles, basal compartment Rab11+vesicles, LMAN1 redistribution, and avoidance of lysosomal fate inenterocytes similar to that observed previously for cholix A-Btranscytosis. Ultimately, the delivery construct is delivered tolysosome-like structures within a large fraction of cells within thelamina propria, an outcome that would limit its systemic distributionfollowing A-B transcytosis.

Example 35: Macrophages in the Lamina Propria are Activated by theDelivery Construct of SEQ ID NO: 5

After determining that the delivery construct of SEQ ID NO: 5 wasbiologically active in vivo, the cellular target of the deliveryconstruct was investigated using immunofluorescent microscopy.Demonstration of hIL-10 delivery to the lamina propria was shown inmouse jejunum tissue samples along with pSTAT3 induction and immune celllabeling (FIG. 67). At this 40 min time point after oral gavage, hIL-10was observed in the basal region of enterocytes and in linearorganizations within the lamina propria that were consistent withlacteals and/or vascular vessels. Areas adjacent to detected hIL-10demonstrated strong pSTAT3 expression in many cells within the laminapropria and, to much lower levels, in some nuclei of polarizedenterocytes. A moderate proportion of CD3+ cells within the laminapropria were observed to co-localize with pSTAT3: many CD3+ cellspresent within this compartment were not positive for pSTAT3 and therewere many pSTAT3+ cells that were not positive for CD3 (FIG. 134). Usingthe macrophage-specific marker F4/80, however, we observed an extensiveco-localization with pSTAT3 (FIGS. 135 and 136). Additional examples ofF4/80 and pSTAT3 labeling are highlighted in tissue samples obtainedfrom the small intestine (FIG. 137) and colon (FIG. 138) obtainedfollowing gavage dosing of the delivery construct of SEQ ID NO: 5 inmice that was consistent with extensive IL-10 activation of macrophagecell populations within the lamina propria. The results suggest that thedelivery construct of SEQ ID NO: 5 ultimately targeted to CD3+Tlymphocytes following A→B transcytosis, possibly through interactionsdriven by its cholix-derived carrier element, while macrophages wereextensively targeted by the hIL-10 element.

Example 36: Circulating IL-1Ra and Systemic hIL-10 Increases in Responseto Oral Gavage with the Delivery Construct of SEQ ID NO: 5 in Mice

The inflammatory actions of IL-1β are modulated by the presence of aspecific receptor antagonist known as IL-1Ra. Increased serum IL-1Ralevels can be induced by IL-10, which can occur in the absence of apro-inflammatory state. With an appreciation of uncertainties associatedwith how much and when biologically active delivery construct isreleased from the stomach to reach the small intestine as well as thetiming of its uptake and actions within the lamina propria followingoral gavage, we examined IL-1Ra to function in non-inflamed mice as abiomarker of the IL-10 delivery construct's actions.

Oral gavage of 10 mg/kg of the delivery construct of SEQ ID NO: 5resulted in ˜50 pM hIL-10 being detected in systemic circulation asearly as 1 h, which increased to ˜700 pM at 4 h (FIG. 139). Detection ofhIL-10 in distal small intestinal (FIG. 140) and colonic (FIG. 141)tissues were not significantly elevated above baseline at 1 and 2.5 hbut showed slight increases with high variability at 4 h; hIL-10 insmall intestinal lysates were ˜10-fold higher than colonic lysates.Serum IL-1Ra levels were significantly increased at 4 h (FIG. 142).These results demonstrated that the onset of IL-1Ra induction occursbetween 2.5-4 h post oral gavage, about the same time of a detectableincrease in tissue levels of hIL-10 was observed in distal smallintestinal and colonic tissues. This result suggests that either therewas a nearly immediate induction of IL-1Ra following the uptake of thedelivery construct in distal small intestinal and colonic tissues, orthat this induction occurred several hours following the nearly rapiduptake of the delivery construct in the upper small intestine after oralgavage. To test this question, the delivery construct was directlydeposited onto the luminal surface of colonic intestinal mucosa in anonhuman primate model.

Example 37: Induction of IL-1Ra Following Intracolonic Spray with theDelivery Construct of SEQ ID NO: 5 in Nonhuman Primates (NHPs)

A topical spraying approach was used to administer 1, 3, or 10 mg (totaldose) of the delivery construct of SEQ ID NO: 5 onto the luminal surfaceof colonic mucosae (proximal, mid, and distal) of healthy, fastednonhuman primates (cynomolgus monkeys). This was achieved using acolonoscope with an associated spray nozzle. In this way, refinedinformation for onset and duration of IL-1Ra induction could be obtainedat specific local doses. Circulating levels of IL-1Ra increased between2-3 h after colonic spray in a manner that suggested a near saturationof this response at the lowest dose tested (FIG. 147). Analysis ofpSTAT3 induction in colon tissues biopsies showed the anticipated timecourse of onset and recovery to baseline for this event that correlatedwith ˜15 min for the delivery construct of SEQ ID NO: 5 to reach thelamina propria following luminal application (FIG. 148). The variabilityof pSTAT3/total STAT3 detected was consistent with tissue capture andstability challenges in this in vivo model. Measurement of tissuedelivery construct levels had similar tissue capture and stabilitychallenges, showing low and variable amounts in the tissue samplescollected (FIG. 143). Importantly, while the delivery construct of SEQID NO: 5 could be detected in colonic tissue, serum levels of thedelivery construct were below the assay detection limit (FIG. 145),consistent with retention of this material in the intestinal laminapropria. Since IL-10 can induce cells to produce more IL-10, we alsolooked for induced IL-10. Within 15 min after an intracolonic spray withthe delivery construct of SEQ ID NO: 5, dose-dependent and transientincreases in tissue IL-10 levels were observed (FIG. 144). Serum levelsof IL-10 were also increased in a dose-dependent manner to the amount ofthe delivery construct dosed by colonic spray (FIG. 146); theseincreased serum levels, while remaining in the picomolar range, weremore durable than those observed in colonic tissue. It is important tonote, due to the similarity of human and NHP IL-10, that ELISA valuesshown could also include hIL-10 that has somehow separated from thecarrier domain of the delivery construct molecule. While this cannot beruled out, no other data supports this as an outcome and it appearslikely that the majority, if not all, tissue and serum IL-10 measuredrepresents an NHP source.

Example 38: Salt Screening for Dimer Stability During Processing

A screen of various salts and salt concentrations was conducted toassess the stability of the IL-10 delivery construct (SEQ ID NO: 5) invarious liquid media. Formulations were prepared with the deliveryconstruct at 20 g/Lin 10 mM Sodium Phosphate at pH 7.0 with the saltsand salt concentrations as indicated in Table 53. The percentage of theIL-10 delivery construct present in dimer form, monomer form, and highmolecular weight aggregates (HMW Agg) was assessed by size exclusionHPLC as indicated previously. Table 53 shows the results immediatelyafter formulating and after a 2-day incubation at 25° C. Table 54 showsresults for further salt concentrations at the initial time point Of theformulations assessed, 1×PBS, 150 mM, and 200 mM NaCl showed the moststability. Higher concentrations were considered for Na₂SO₄ and NH₄SO₄(data not shown). However, the IL-10 delivery construct was seen toprecipitate during buffer exchange.

TABLE 53 IL-10 delivery construct (SEQ ID NO: 5) dimer percentage indifferent salt formulations t0 25 C., 2 days Formulation HMW Agg DimerMonomer HMW Agg Dimer Monomer Starting material 3.5 88.8 7.7 5.1 86.768.2 NaCl 150 mM 3.7 91.3 5.0 6.3 83.9 9.8 NaCl 200 mM 2.8 91.7 5.4 5.784.3 9.97 NaCl 250 mM 3.6 91.4 4.97 5.5 84.3 10.1 KCl 50 mM 3.5 91.6 4.97.0 84.1 8.9 KCl 100 mM 3.9 91.5 4.6 7.8 81.5 10.6 KCl 150 mM 3.1 91.65.3 6.3 83.9 9.8 MgCl₂ 25 mM 9.6 85.6 4.8 40.8 52.8 6.4 MgCl₂ 50 mM 8.587.2 4.3 31.9 60.5 7.5 MgCl₂ 75 mM 7.2 86.2 6.6 23.0 67.9 9.0 Na₂SO₄ 250mM 3.3 90.9 5.7 5.0 82.5 12.5 Na₂SO₄ 500 mM 4.2 90.9 4.9 6.7 83.4 9.9NH₄SO₄ 250 mM 3.2 89.6 7.1 6.1 79.6 14.3 NH₄SO₄ 500 mM 3.5 89.7 6.8 8.574.1 17.4

TABLE 54 IL-10 delivery construct (SEQ ID NO: 5) dimer percentage indifferent salt formulations Buffer Salt Concentration HMW Agg DimerMonomer starting material 3.1 92.3 4.6 10 mM Sodium NaCl 150 mM 3.3 91.84.9 Phosphate 500 mM 2.7 92.0 5.2 1M 2.8 91.6 5.6 Na₂SO₄  25 mM 3.1 91.75.2  50 mM 2.9 91.9 5.1  75 mM 3.4 91.4 5.2 NH₄SO₄  25 mM 4.1 90.5 5.4 50 mM 2.9 91.9 5.2  75 mM 3.2 91.5 5.3 100 mM 3.2 91.0 5.8

Example 39: Comparison of Different Purification Conditions with S-650FCation Exchange Columns for an IL-10 Delivery Construct

IL-10 delivery construct (SEQ ID NO: 5) was purified from inclusionbodies using a process analogous to that described in FIG. 2B. Refoldingwas carried out at 4° C. in a refolding solution containing 1 mM reducedglutathione, 0.5 mM oxidized glutathione, 1M Arginine-HCl, 250 mMsucrose, 100 mM Tris pH 8.5 at 4° C., and 2 μm EDTA. IL-1 deliveryconstruct (SEQ ID NO: 5) was purified using each of the three processtrains described in Tables 55-57. Buffer A for the CHT bind/elute stepin Table 56 may also comprise 25 mm Tris, pH7.5, 300 mM NaCl, 5.0 mMNaPi, 0.5 mM CaCl2. Columns used are described in Table 58.

TABLE 55 Process train 1 Buffer Buffer Sample Column Column Column A Bprep equilibration wash Elution Capto Q 25 mM Tris 25 mM Tris 2 × UFDF15% B  15% B 15-40% B, ImpRes pH 7.5 pH 7.5, 1M (25 mM 16 CV NaCl TrispH 7.5, 150 mM NaCl) S-650F 20 mM NaPi 20 mM NaPi 1/1 Dilution 20% B 20% B 20-45% B, pH 7.0 pH 7.0, 1M with 40 mM 10 CV NaCl NaPi pH 7.0 CHT80 mM NaPi 200 mM Add 1M  0% B 100% B FT-mode (FT- pH 7.0 NaPi pH 7.0NaPi pH 7.0 mode) to a final of 60 mM (X L × 0.0417)

TABLE 56 Process train 2 Buffer Buffer Sample Column Column Column A Bprep equilibration wash Elution Capto Q 25 mM 25 mM 2 × UFDF (25 15% B15% B 15-40% B, ImpRes Tris pH 7.5 Tris mM Tris 16 CV pH 7.5, pH 7.5,150 gradient 1M NaCl mM NaCl) elution CHT 25 mM 200 mM none  0% B  0% B30% B 2CV (Bind/Elute) Tris NaPi Step elution pH 7.5, pH 7.0 150 mM NaClS-650F 20 mM 20 mM Dilute 1:1 20% B 20% B 20-45% B, NaPi NaPi with water10 CV pH 7.0 pH 7.0, gradient 1M NaCl elution

TABLE 57 Process train 3 Buffer Buffer Sample Column Column Column A Bprep equilibration wash Elution S-650F 20 mM 25 mM 2 × UFDF (25 20% B 20% B Step NaPi pH 7.0 NaPi (NaPi gradient pH 7.0, 1M pH 7.0, (45% B),NaCl 200 mM) 2-3 CV Capto Q 25 mM Tris 25 mM Tris Dilute 1/2 15% B  15%B 15-40% B, ImpRes pH 7.5 pH 7.5, 1M with 75 16 CV NaCl mM Tris pH 7.5CHT 40 mM 200 mM Add 1M  0% B 100% B FT-mode (FT- NaPi pH 7.0 NaPi pH7.0 NaPi mode) pH 7.0 to a final of 60 mM (X L × 0.064)

TABLE 58 Columns Starting amount Column Column DBC (5 L refold) volumeCapto Q ImpRes 16 g/L 5 g (20 cm h) 320 mL S-650F 20 g/L 200 mg (20 cmh)  41 mL CHT 20 g/L 200 mg (20 cm h) 106 mL

The yield, recovery, and purity of the IL-10 delivery construct wasassess after each step, and after each process train. Table 59 shows acomparison between process train 2 and a control process.

TABLE 59 comparison between process train 2 (PT2) and control NameProcess Yield Purity Recovery PT2 Capto Q ImpRes 26 mg 78.5%   1% CHTstep B/E 80 mM NaPi S-650F Control Capto Q ImpRes 68 mg 78.2    1.5% CHTgradient B/E

TABLE 60 comparison of different process trains Process TrainChromatography Purity Recovery Endotoxin Control* Capto Q *85%  —  *8EU/mg CHT (Linear elution) PT1 Capto Q 92% 24% 8.9 EU/mg S-650F (FTmode) CHT PT2 Capto Q 91% 24%   5 EU/mg CHT (Step elution) S-650F PT3S-650F 96% 20% 0.4 EU/mg Capto Q CHT (FT mode) *Based on historical data

Table 60 shows a comparison of the three different process trains, and acontrol process train without an S-650F column. All three process trainswith S-650F cation exchange column purifications resulted in higherpurity of the IL-10 delivery construct. Process trains 2 and 3 resultedin lower levels of endotoxin than the control process. It was surprisingthat a cation exchange resin was useful in the purification of the IL-10delivery construct as cation exchange resins are negatively charged, andthus bind to proteins with a positive charge. Here, the IL-10 deliveryconstruct has a calculated isoelectric point (pI) of 5.49, and thus isanticipated to have a negative charge under conditions traditionallyused in a cation exchange chromatography. Surprisingly, however, theIL-10 delivery construct bound to the cation exchange resin.

A possible explanation for the IL-10 delivery construct binding to theSulfate-650F resin is that, while the calculated pI for the IL-10delivery construct is 5.49, each domain (cholix and IL-10) maintains itsown unique properties and has a local pI that differs significantly fromthe pI of the overall construct. For instance, the calculated p1 ofIL-10 is 8.1 and under working conditions (e.g., pH 7) the IL-10delivery construct would have a positively charged IL-10 domain thatbinds to the cation exchange resin.

Example 40: A Randomized Double-Blind Placebo Controlled ExploratoryMedicine Trial in Adults with Active Rheumatoid Arthritis Who haveDemonstrated an Inadequate (Partial) Response to Anti-TNF Therapy

IL-10 is considered a master regulator of the innate and adaptive immunesystem as it inhibits not only the inflammasome but many inflammatoryevents found to be associated with RA beginning with macrophageactivation and secretion of IL-1, IL-6, TNF alpha, MMP-1/2 whilereducing systemic signs of inflammation and development of T regulatorycells.

Oral delivery of an IL-10 delivery construct provides local delivery ofIL-10 to the GI mucosal immune system while avoiding high systemiclevels of the drug. Providing local delivery of IL-10 to the GI mucosamay also be able to effect systemic immunoregulation. Modulation ofimmune cell activity as the immune cells traffic through the GI mucosamay result in effective systemic immunoregulation as those cells thenmove throughout the body. Thus, targeted delivery of IL-10 to the GImucosal immune system may be able to cause effective immunoregulationfor treatment of, for example, rheumatoid arthritis subjects withenhanced safety.

This study assesses the safety and tolerability of 12 weeks of dailyoral IL-10 via an IL-10 delivery construct in subjects with activerheumatoid arthritis who have demonstrated only a partial response toTNF inhibitors. The study also assesses the biologic activity of 12weeks of daily oral treatment with an IL-10 delivery construct bychanges in the Disease Activity Score 28 (DAS-28) (CRF), SimplifiedDisease Activity Index (SDAI) and Clinical Disease Activity Index(CDAI). Additionally, the study examines the pharmacokinetic andpharmacodynamic effects of the IL-10 delivery construct and to examinethe immunogenicity of the IL-10 delivery construct. The study alsoevaluates quality of life (QOL) with Health Assessment Questionnairewithout Didability Index (HAQ-DI ) after 12 weeks of daily oral IL-10via the IL-10 delivery construct.

This is a randomized double-blind placebo-controlled study inapproximately 18 subjects with active Rheumatoid Arthritis who had aninadequate (partial) response to anti-TNF therapy. Subjects arerandomized in 2:1 ratio to receive either the IL-10 delivery constructor placebo for 12 weeks. The study population comprises adult subjectswith active Rheumatoid Arthritis who had an inadequate (partial)response to anti TNF therapy.

Subjects are included in the study if they fit the following inclusioncriteria. Subjects are aged ≥18 and <75 years old at the time ofinformed consent. Subjects have a diagnosis of rheumatoid arthritis (RA)under the 1987 American College of Rheumatology (ACR) or 2010ACR/European League Against Rheumatism (EULAR) criteria. Subjects havereceived anti TNF biologic treatment under approved dosage andadministration for ≥12 weeks but only had a partial response. A historyof biologics treatment should be limited to 1 or 2 anti TNF agents amongadalimumab, infliximab, golimumab, etanercept, (including biosimilars).Subjects have at least one joint showing active disease by MRI/FOI andtwo or more tender joints (out of 28 joints) and 2 or more swollenjoints in the Screening Phase. Subjects are able to continue stable doseregimen of anti-TNF until completion or until study discontinuation.Subjects have a C-reactive protein (CRP) level ≥0.6 mg/deciliter (dL) orerythrocyte sedimentation rate (ESR) ≥28 millimeters per hour (mm/hr.)in the Screening Phase. Subjects have a weight of ≥30 kilograms (kg) and≤100 kg. Subjects are required to voluntarily consent, in writing, toparticipate in this study. All subjects are thoroughly briefed on theconditions for participation in the study, is able to understand, andmust be willing and able to comply with all aspects of the protocol.

Subjects are excluded from the study if they have any medical historywhich may suggest increased risk or possible confounding factors.Subjects are excluded from the study if they have an inflammatoryarthritic disorder other than rheumatoid arthritis or Sjogren'ssyndrome, or if they are diagnosed with rheumatoid arthritis class IV(according to ACR 1991 Revised Criteria for the Classification of GlobalFunctional Status). Subjects are also excluded if they have recentlyreceived an immunoglobulin preparation, blood products or a livevaccine. Subjects are excluded from this study if they have a history ofsevere allergy (shock or anaphylactoid symptoms). Subjects are alsoexcluded if they have a history of, or current condition of, cancer, orimmunodeficiency. Subjects with a history of, or current, infectiousdisease may also be excluded. Subjects must not be pregnant orlactating, and must agree to use a highly effective method ofcontraception for the duration, and for some time after, the trial.Subjects are excluded from this study if they have any history of amedical condition or a concomitant medical condition that in the opinionof the investigator or sub-investigator would compromise theparticipant's ability to safely complete the study

Outcome Measures

Subjects are monitored clinically, including by physical exams,hematology, chemistry, EKG, and urinalysis. Clinical efficacy isassessed by several different measures. Disease Activity Score 28(DAS-28) is a mathematically calculated, continuous, composite endpointwith differential weighting given to each of the following components:tender joint count (28 joints), swollen joint count (28 joints), acutephase reactant, and patient global assessment of arthritis. SimplifiedDisease Activity Index (SDAI) measures the number of swollen and tenderjoints (shoulder, elbow, wrist, metacarpal phalangeal joints andproximal phalangeal joints of the hand), Patient and Physician globalassessments of Disease Activity and CRP. CDAI is the same as SDAI butomits the CRP value. HAQ-DI assesses the degree of difficulty a patienthas experienced during the past week in eight domains of daily livingactivities: dressing and grooming, arising, eating, walking, hygiene,reach, grip, and other activities. DAS28 (case report form), SDAI andCDAI, are all assessed at baseline, weeks 2, 4 8; and 12; HealthAssessment Questionnaire-Disability Index (HAQ-DI) to assess physicalfunction changes will be assessed at baseline and weeks 4, 8, 12 and 16(4 weeks after last dose).

Serum levels of the IL-10 delivery construct and IL-10 are assessed atbaseline and Day 1 at 4, 8 and 24 hrs. post dose and at weeks 2, 4, 8,12 and 16.

Pharmacodynamic markers that are assessed include monocyte HLA-DRexpression; serum IL-1Ra, CRP, Serum Amyloid A, VCAM-1, IL-1, IL-6, TNFalpha, MMP1/2, ESR, EGF and VEGF-alpha: obtained at baseline and Day1-at 24 hrs. and weeks 2, 4, 8 12 and 16. The total/titer of anti-IL-10delivery construct levels is assessed at weeks 12 and 16.

Subject safety will be monitored and subjects are removed from the studyif they experience adverse events or serious adverse events, orclinically significant laboratory changes.

Treatment efficacy is considered both in terms of change over time, andat completion of therapy and 4 weeks post completion in:

1. DAS-28 (case report form), SDAI and CDAI;2. Proportion of subjects with DAS-28 less than 2.6;3. HAQ-DI compared to baseline; and4. Change in acute phase reactants (CRF, ESR).

Changes in the IL-10 delivery construct and serum IL-10 levels comparedto baseline: Day 1-8 and 24 hrs. and at weeks 2, 4, 8 and 12 is plottedand considered. Changes in biomarkers are compared to baseline formonocyte HLA-DR expression, serum IL-1Ra, Serum Amyloid A, VCAM-1, IL-1,IL-6, TNF alpha, MMP1/2, EGF and VEGF-alpha: baseline and Day 1-at 8 and24 hrs. and weeks 2, 4, 8 and 12. The incidence of anti-drug antibodiesat weeks 12 and 16 is compared to baseline. A positive outcome may be anincremental benefit in any of the abovementioned parameters with thecombination therapy as compared to the TNF alpha inhibitor alone.

Example 41: Exploratory Medical Trial-Ulcerative Colitis TNF PartialResponders

This study aims to evaluate the efficacy and safety of an IL-10 deliveryconstruct in patients with moderate to severe active ulcerative colitiswho are partial responders to anti-Tumor Necrosis Factor α antibodytreatments.

This study assesses the safety and tolerability of 12 weeks of dailyIL-10 via an oral IL-10 delivery construct in subjects with moderate tosevere ulcerative colitis who have demonstrated only a partial responseto TNF monoclonal antibody inhibitors. The study also assesses theclinical activity of 12 weeks of daily IL-10 via an oral IL-10 deliveryconstruct by changes in the Modified Mayo Score, MMS subscales andhistopathology. This study examines the pharmacokinetic andpharmacodynamic effects of the IL-10 delivery construct, as well as it'simmunogenicity.

This is an open label study in approximately 12 subjects with moderateto severe Ulcerative Colitis who had an inadequate (partial) response toanti-TNF therapy. Subjects receive an IL-10 delivery constructcorresponding to the amino acid sequence of SEQ ID NO: 5 for 12 weekswhile continuing their stable dose of anti-TNF therapy and are followedfor safety for an additional 4 weeks.

The study population is drawn from adult subjects with active moderateto severe Ulcerative Colitis (Modified Mayo Score of 4-9 excluding GPAand with a central read Mayo endoscopic sub score of 2 or 3) who had aninadequate (partial) response to anti-TNF monoclonal antibody therapy.

Potential subjects are assessed by the following eligibility criteria.For inclusion all patients must provide written informed consent; and bebetween 18-75 years.

Subjects have a diagnosis of UC according to American College ofGastroenterology guidelines. Subjects have moderate-to-severe active UC,at time of screening, defined as: Modified Mayo Clinic Score (MMS) ofbetween 4-9 points AND a centrally read MCS endoscopic sub score ofgrade 2 or higher, AND MMS rectal bleeding sub score of 1 point orhigher, AND disease extending 15 cm or more from the anal verge. Stabledoses of allowed concomitant medications include: stable oralcorticosteroids (i.e., <20 mg/day of prednisone, <9 mg/day ofbudesonide) ≥2 weeks before D1 dosing; tapering of oral corticosteroidsper Investigator's discretion during the study is allowed; stable oral5-amyinosalicylic acid dose ≥2 weeks before D1 dosing; stable doses ofprobiotics ≥2 weeks before D1 dosing; and stable anti-diarrheas ≥2 weeksbefore D1 dosing. Patients must be receiving anti-tumor necrosis factoralpha therapy for UC and have demonstrated an inadequate (partial)response before D1 dosing or must be naïve to anti-TNF therapy prior toscreening; allowed anti-TNFs include Infliximab (Remicade), adalimumab(humira) and golimumab (Simponi) but excluding etanercept

Patients previously treated with cyclosporine or tacrolimus must havediscontinued therapy ≥4 weeks before D1 dosing. Topical corticosteroidsand topical 5-amyinosalicylic acid preparations must have been withdrawn≥2 weeks before D1 dosing. Nonsteroidal anti-inflammatory drugs (NSAIDs)must have been discontinued ≥4 weeks before D1 dosing. Tofacitinib orother Janus kinase (JAK) inhibitors must have been discontinued ≥2 weeksbefore D1 dosing. Females with reproductive potential must have anegative pregnancy test result before enrollment. Men and women withreproductive potential have to be willing to use a highly effectivemethod of contraception from study start to ≥3 months after the finaldose of the study drug. A highly effective method of birth control isdefined as one which results in a low failure rate (less than 1% peryear).

Subjects are excluded from this study if they have any of the followingGI related exclusion criteria: indeterminate colitis (IBD-U) orsuspected Crohn's disease, any history of colectomy, presence of anileostomy or colostomy, a history or evidence of colonic mucosaldysplasia or short gut syndrome.

Subjects are excluded from this study if they have any of the followinggeneral health related exclusion criteria: pregnant or lactating,inability to comply with study protocol in the opinion of theinvestigator, history of dysplasia or malignancy in recent 5 years,except completely excised basal cell carcinoma or squamous cellcarcinoma of the skin or carcinoma in situ of the cervix, cirrhosis oractive alcohol abuse per the judgement of investigator, poorlycontrolled diabetes (HbA1c>8.0%), significant screening ECGabnormalities, or impaired renal function. Subjects with evidence ofcurrent or previous clinically significant disease, medical condition orfinding in the medical examination that in the opinion of theinvestigator, would compromise the safety of the patient or quality ofthe data are excluded from this study.

Subjects are monitored clinically, including by physical exams,hematology, chemistry, EKG, and urinalysis. Clinical efficacy isassessed by several different measures. The proportion of patients withclinical remission at week 12 is assessed. Clinical remission is definedas an endoscopic sub score of 0/1, a rectal bleeding sub score of 0, anda stool frequency sub score of 0 or 1 with at least a 1-point reductionfrom baseline. Clinical remission may also be defined as: stoolfrequency=0; stool bleeding=0; endoscopy score of 0 or 1 stool frequencysub score can also be at least 1-point decrease in stool frequency subscore from baseline (start of trial) to achieve a stool frequency subscore=0 or 1.

The proportion of patients with endoscopic response is assessed.Endoscopic response is defined as a Modified Mayo Clinical Scoreendoscopic subscale score of 0 or 1. Changes of histological activitygrade from baseline using the Geboes or Robarts Histopathology Indexsystem will be assessed. Histological healing is defined as histologicalgrade=0.

Serum levels of the IL-10 delivery construct and IL-10 are assessed atbaseline and at Day 1, weeks 2, 4, 8 and 12.

Pharmacodynamic endpoints which are assessed include monocyte HLA-DRexpression; serum IL-1Ra, CRP, ESR, fecal calprotectin changes, IL-1,IL-6, TNF alpha, MMP1/2, and IFNg obtained at baseline and Day 1 andweeks 2, 4, 8, 12 and 16.

The total/titer of anti-IL-10 delivery construct levels is assessed atbaseline and weeks 8, 12 and 16. Biopsies of colon at baseline and atend of treatment phase examined by light microscopy,immunohistochemistry, flow cytometry and gene arrays.

Subject safety is monitored, and subjects are removed from the study ifthey experience adverse events or serious adverse events, or clinicallysignificant laboratory changes.

Treatment efficacy is considered both in terms of change over time, andat 4, 8 and 12 weeks and 4 weeks post completion in:

1. Change in Modified Mayo Score;

2. Change in Stool Bleeding subscale;3. Change in Stool Frequency subscale;4. Change in endoscopy subscale;5. Change in acute phase reactants (CRF, ESR); and6. Change in histopathology score (Geboes or Robarts).

Changes in the IL-10 delivery construct and serum IL-10 levels comparedto baseline: Day 1-8 and 24 hrs and at weeks 2, 4, 8 and 12 are plottedand considered. Changes in biomarkers compared to baseline areconsidered for: monocyte HLA-DR expression serum IL-1Ra, IL-1, IL-6, TNFalpha, MMP1/2, EGF and VEGF-alpha at baseline and Day 1-at 8 and 24 hrs.and weeks 2, 4, 8 and 12. The incidence of anti-drug antibodies at weeks12 and 16 is compared to baseline. Specific stains will be used tovisualize infiltrating inflammatory cells and HLA-DR expression incolonic epithelium by Immunochemistry and/or flow cytometry and genearray. A positive outcome may be an incremental benefit in any of theabovementioned parameters with the combination therapy as compared tothe TNF alpha inhibitor alone.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. (canceled)
 2. A solid pharmaceutical composition suitable for oraladministration to a subject in need thereof, the pharmaceuticalcomposition comprising: a plurality of delivery constructs; and one ormore pharmaceutically acceptable excipients; wherein each of thedelivery constructs comprises an amino acid sequence with at least 90%sequence identity to SEQ ID NO: 5 or SEQ ID NO: 13; wherein at least 80%of the delivery constructs are in a dimer form; wherein thepharmaceutical composition is formulated as an oral dosage form, theoral dosage form comprising (1) a first coat comprising two or morecopolymers each having a different dissolution pH and (2) another coatinterior to the first coat and exterior to the delivery constructs andthe one or more pharmaceutically acceptable excipients; and wherein afirst copolymer of the first coat comprises a polymer of formula I:

wherein x, y, and n in Formula I are each greater than or equal to one;and wherein a second copolymer of the first coat comprises a copolymerof formula II:

wherein x, y, z, and n in Formula II are each greater than or equal toone.
 3. The pharmaceutical composition of claim 2, wherein each of thedelivery constructs comprises an amino acid sequence with at least 95%sequence identity to SEQ ID NO: 5 or SEQ ID NO:
 13. 4. Thepharmaceutical composition of claim 2, wherein the one or morepharmaceutically acceptable excipients does not include a polysorbate.5. The pharmaceutical composition of claim 2, wherein the one or morepharmaceutically acceptable excipients comprises a non-ionic lubricant.6. The pharmaceutical composition of claim 5, wherein the non-ioniclubricant is glyceryl behenate.
 7. The pharmaceutical composition ofclaim 2, wherein the one or more pharmaceutically acceptable excipientsdoes not include a magnesium stearate.
 8. The pharmaceutical compositionof claim 2, wherein the pharmaceutical composition is configured torelease substantially none of the plurality of delivery constructs aftera 1-hour exposure to a solution having a pH of 1.0 in a Type 4dissolution apparatus in open mode, wherein the solution having the pHof 1.0 is a dissolution media containing hydrochloric acid.
 9. Thepharmaceutical composition of claim 8, wherein the pharmaceuticalcomposition is configured to release at least 40% of the deliveryconstruct after 2 hours of exposure to a solution having a pH of 7.0 ina Type 4 dissolution apparatus in open mode, wherein the solution havingthe pH of 7.0 is a citrate/phosphate buffer.
 10. The pharmaceuticalcomposition of claim 2, wherein a ratio of free carboxyl groups to estergroups in the first copolymer is from 0.8:1 and 1.2:1 and wherein aratio of free carboxyl groups to ester groups in the second copolymer isfrom 0.8:1 to 1.2:1.
 11. The pharmaceutical composition of claim 2,wherein a ratio of the first copolymer of the first coat to the secondcopolymer of the first coat is from 15:85 to 55:45.
 12. Thepharmaceutical composition of claim 2, wherein the ratio of the firstcopolymer to the second copolymer is to 30:70.
 13. The pharmaceuticalcomposition of claim 2, wherein the first coat further comprises ananti-tacking agent, a plasticizer, a surfactant, or a combinationthereof.
 14. The pharmaceutical composition of claim 2, furthercomprising an additional coat exterior to the first coat.
 15. Thepharmaceutical composition of claim 14, wherein the additional coatexterior to the first coat comprises hydroxypropyl methylcellulose(HPMC).
 16. The pharmaceutical composition of claim 2, wherein theanother coat comprises HPMC.
 17. The pharmaceutical composition of claim2, wherein the oral formulation is in a tablet or a capsule form. 18.The pharmaceutical composition of claim 2, wherein the oral formulationis in a unit dose form.
 19. The pharmaceutical composition of claim 18,wherein the first coat has a mass from 10 mg to 100 mg per unit doseform.
 20. The pharmaceutical composition of claim 18, wherein theplurality of delivery constructs are present in an amount from 1 mg to20 mg per unit dose of the formulation.
 21. A method of treating aninflammatory disease in a subject, the method comprising administeringto the subject an effective amount of an oral formulation comprising thepharmaceutical composition of claim
 2. 22. The method of claim 21,wherein the inflammatory disease is ulcerative colitis, proctitis,pouchitis, Crohn's disease, multiple sclerosis (MS), systemic lupuserythematosus (SLE), graft versus host disease (GVHD), rheumatoidarthritis, inflammatory bowel disease (IBD), celiac disease, psoriaticarthritis, or psoriasis.
 23. The method of claim 22, wherein theinflammatory disease is ulcerative colitis.
 24. The method of claim 22,wherein the inflammatory disease is rheumatoid arthritis.
 25. The methodof claim 22, wherein the inflammatory disease is pouchitis.
 26. A methodfor producing a pharmaceutical composition for oral administration thatcomprises a plurality of coatings, the method comprising: obtaining adelivery construct that comprises an amino acid sequence with at least90% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 13; applying aplurality of coatings around the delivery construct, wherein theplurality of coatings comprises (1) a first coating with two or morecopolymers each having a different dissolution pH and (2) anothercoating interior to the first coating and exterior to the deliveryconstruct; wherein a first copolymer of the first coating comprises apolymer of formula I:

wherein x, y, and n in Formula I are each greater than or equal to one;and wherein a second copolymer of the first coating comprises acopolymer of formula II:

wherein x, y, z, and n in Formula II are each greater than or equal toone.
 27. The method of claim 26, wherein the delivery constructcomprises an amino acid sequence with at least 95% sequence identity toSEQ ID NO: 5 or SEQ ID NO:
 13. 28. The method of claim 26, wherein atleast 80% of the delivery construct in the pharmaceutical formulation isin a dimer form.
 29. The method of claim 26, wherein the pharmaceuticalcomposition is a solid pharmaceutical composition.
 30. The method ofclaim 29, wherein the method further comprises lyophilizing orspray-drying of the delivery construct.
 31. The method of claim 26,further comprising adding of one or more pharmaceutically suitableexcipients to the pharmaceutical composition.
 32. The method of claim31, further comprising compacting the delivery construct with the one ormore pharmaceutically suitable excipients.